Genomic DNA encoding the pseudomonas fluorescens alternative sigma factor, RpoS, capable of activating gene expression

ABSTRACT

Gene activating sequences which activate the expression of other bacterial genes, which are latent or expressed at low levels, are provided. The gene activating sequences confer the ability to produce several metabolites and may be transferred to bacterial strains. The transformed biocontrol agents are active to inhibit the growth of the fungal pathogens.

This is a divisional application of Ser. No. 08/287,442, filed Aug. 8,1994, which is a continuation-in-part of Ser. No. 08/258,261, filed Jun.8, 1994, now U.S. Patent No. 5,639,949, which is a continuation-in-partof Ser. No. 08/087,636, filed Jul. 1, 1993, now abandoned which is acontinuation-in-part of Ser. No. 07/908,284, filed Jul. 2, 1992, nowabandoned which is a continuation-in-part of Ser. No. 07/570,184, filedAug. 20, 1990, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the identification, isolation, cloningand use of genetic elements which contribute to the activation of genesin bacterial strains. More specifically, the invention relates to theidentification of three classes of genetic elements which interact witheach other in the activation of genes in bacteria. Manipulation of thesetypes of elements, either separately or in combination, can be used tomanipulate bacterial phenotype.

BACKGROUND OF THE INVENTION

It has been recognized that crops grown in some soils are naturallyresistant to certain fungal pathogens. Furthermore, soils that areconducive to the development of these diseases can be renderedsuppressive, or resistant, by the addition of small quantities of soilfrom a suppressive field. Scher et al. Phytopathology 70:421 (1980).Conversely, suppressive soils can be made conducive to fungal diseasesby autoclaving, indicating that the factors responsible for diseasecontrol are biological. Subsequent research has demonstrated that rootcolonizing bacteria are responsible for this phenomenon known asbiological disease control (BDC). Baker et al., Biological control ofplant pathogens, (Freeman Press, San Francisco)(1974).

In many cases, the most efficient strains of biological diseasecontrolling bacteria are fluorescent Pseudomonads. Weller et al.,Phytopathology, 73:463-469 (1983). These bacteria have also been shownto promote plant growth in the absence of a specific fungal pathogen bythe suppression of detrimental rhizosphere microflora present in mostsoils. Kloepper et al., Phytopathology 71:1020-1024 (1981). Importantplant pathogens that have been effectively controlled by seedinoculation with these bacteria include Gaemannomyces gramminis, thecausative agent of take-all in wheat, Cook et al., Soil Biol. Biochem8:269-273 (1976) and Pythium and Rhizoctonia, pathogens involved indamping off of cotton. Howell et al., Phytopathology 69:480-482 (1979).Rhizoctonia is a particularly problematic plant pathogen for severalreasons. First, it is capable of infecting a wide range of crop plants.Second, there are no commercially available chemical fungicides that areeffective in controlling the fungus. Because of these circumstances, aninhibitor against R. solani would be of substantial interest as apotential control for this pathogen.

Many biological disease controlling Pseudomonas strains produceantibiotics that inhibit the growth of fungal pathogens. Howell et al.,Phytopathology 69:480-482 (1979); Howell et al. Phytopathology70:712-715 (1980). These have been implicated in the control of fungalpathogens in the rhizosphere. Several past studies have focused on theeffects of mutations that result in the inability of the disease controlbacterium to synthesize these antibiotics. Kloepper et al.,Phytopathology 71: 1020-1024 (1981); Howell et al., Can. J. Microbiol.29:321-324 (1983). In these cases, the ability of the organism tocontrol the pathogen is reduced, but not eliminated. In particular,Howell et al., Phytopathology 69:480-482 (1979) discloses a strain ofPseudomonas fluorescens which was shown to produce an antibioticsubstance that is antagonistic to Rhizoctonia solani.

In Baker et al., Biological Control of Plant Pathogens, (AmericanPhytopathological Society, St. Paul, Minn.)(1982), pages 61-106, it isreported that an important factor in biological control is the abilityof an organism to compete in a given environment. Thus, it is desirableto obtain strains of biocontrol agents which are effective to controlthe growth of fungal pathogens, such as Rhizoctonia solani,Helminthosporium gramineae and species of the genera Pythium andFusarium and are able to aggressively compete with indigenous bacteriaand microflora that exist in the rhizosphere of the plant. In order toachieve this objective, it is further desirable to obtain DNA sequenceswhich are useful in conferring resistance to fungal pathogens which maybe used to genetically engineer strains of biocontrol agents thatcombine the ability to control the growth of fungal pathogens with theability to control other plant pathogens and/or the ability toaggressively compete in the rhizosphere.

Bacterial two-component regulatory systems have been extensivelyreviewed (e.g. Albright et al., Annu. Rev. Genet. 23:311-336 (1989);Bourret et al., Annu. Rev. Biochem 60:401-441 (1991); Mekalanos, J.Bacteriol. 174:1-7 (1992)). In most instances, an environmental signalis received by a sensor protein component. Reception of the signalinduces an autophosphorylation event and a change in the conformation ofthe sensor protein. In this new conformation, the sensor is capable ofphosphorylating the amino-terminal portion of the activator proteincomponent. This phosphorylation event is thought to alter theconformation of the activator protein such that the DNA binding moduleof its carboxy-terminal end is capable of interacting with the promoterregions of regulated genes within the network.

Laville et al., PNAS:USA 89:1562-1566 (1992) disclose the isolation fromPseudomonas fluorescens CHA0, an activator-component gene designatedgacA which was required for the expression of genes involved in thesynthesis of the antifungal secondary metabolites2,4-diacetylphloroglucinol, cyanide, and pyoluteorin. The strain wasindicated to be capable of suppressing black root rot of tobacco causedby the fungal pathogen Thielaviopsis basicola. Disruption of the gacAgene resulted in a mutant unable to synthesize any of these secondarymetabolites and significantly reduced in its ability to suppress blackroot rot. Laville et al. concluded that the gacA gene was involved inregulation of secondary metabolism in P. fluorescens and inferred thatextracellular secondary metabolites produced under gacA control areimportant for the biocontrol of black root rot. They noted the presenceof a gene homologous to gacA in E. coli and cited preliminary evidencefrom hybridization experiments that a sequence similar to gacA exists inPseudomonas aeruginosa.

Our data indicate that antifungal secondary metabolites are likely onlya subset of factors under the regulatory control of the ORF 5 gene wehave cloned from Pseudornonas fluorescens, since we have discovered thatORF 5, (gafA) also affects the production of hydrolytic enzymes such aschitinase and gelatinase which are involved in the catabolism ofpolymeric carbon sources. Laville et al. did not disclose or suggestthat the gacA gene was involved in activation of latent genes by atranscriptional activator or suggest that an activator derived from onebacterial strain would induce expression of genes from a heterologousbacterium. To our knowledge, the present invention is the first todescribe the unexpected finding that certain natural bacterial isolatescarry undetected, latent genes which can be activated upon introductionof a bacterial transcription activator derived from a different organism(in this case, either upon introduction of a 2.0 kb XhoI fragment of P.fluorescens strain 915 DNA containing ORF 5 or upon introduction of acloned E. coli gene homologous to ORF 5, gafA).

A number of sensory-component genes have been characterized fromdifferent microorganisms (for reviews see Bourret et al., 1991 and Stocket al., 1990). In one case the sensory component gene, referred to aslemA, was found to be required for the pathogenicity of Pseudomonassyringae (Hrabak et al., (1992) J. Bacteriol. 174:3011-3020. However, incontradiction to the data presented in this application, Hrabak et al.proposed that lemA alone may perform sensory and regulatory functions.Our experiments characterize a member of the same gene family clonedfrom Pseudomonas fluorescens and reveal that it plays a crucial role ingene activation by interacting with the global regulatory element gafA.

In Escherichia coli, it has been demonstrated that an alternate sigmafactor designated rpoS is required for the transcription of a large setof genes which are preferentially expressed during stationary phaseconditions (i.e. conditions of nutrient stress) (Siegele and Kolter, J.Bacteriol. 174:345-348 ( 1992); Hengge-Aronis, Cell 72:165-168 ( 1993)). The present invention provides a characterization of a member of therpoS gene family cloned from Pseudomonas fluorescens and reveals itscrucial role in gene activation.

SUMMARY OF THE INVENTION

Accordingly, it is one object of the present invention to provide DNAsequences which are useful in activating genes in bacterial strains.

It is another object of the present invention to provide genes that canbe used to improve the biocontrol capabilities of strains of bacteriaused for biocontrol.

It is one feature of the present invention that DNA sequences and genesare provided that activate genes in bacterial strains.

It is a further feature of the present invention that modified DNAsequences and genes be provided which encode modified proteins, whichenhance the activation of genes in bacterial strains. Such modificationsmay improve the efficacy of regulatory genes.

It is an advantage of the present invention that biocontrol agents maybe produced which are able to inhibit a broad spectrum of plantpathogens.

It is another advantage of the present invention that biocontrol agentsmay be produced which are able to aggressively compete in the plantrhizosphere, which biocontrol agents contain a DNA sequence thatactivates genes in the bacterial biocontrol agent.

According to the present invention, the above objectives may be carriedout by the isolation and use of genetic elements or gene activatingsequences that are able to activate genes that are not normally turnedon in bacterial strains. The isolation of these gene activatingsequences is important for several reasons. First, the activated strainsproduce substances, such as pyrrolnitrin and chitinase, which are ableto inhibit plant pathogens, particularly fungal pathogens, such asRhizoctonia solani, Helminthosporium gramineae and species of the generaPythium and Fusarium. Therefore, use of bacterial strains transformedwith the ORF 5-type, lemA-type, or rpoS-type genetic element (orcombinations of these elements) provides an environmentally safe andeffective method of control of these pathogens.

In addition, these gene activating sequences can be transferred to otherbacterial strains, especially pseudomonad strains, that otherwise arenot effective biocontrol agents for R. solani and thereby transform theminto effective biocontrol strains. The use of the gene activatingsequences to improve the biocontrol capabilities of other strains ofrhizosphere biocontrol strains is also pan of the present invention. Forexample, U.S. Pat. No. 4,456,684, (Weller et al.) discloses thattake-all, a disease of wheat caused by the fungus Gaemannomycesgramminis, can be controlled in some cases by the application ofbacteria inhibitory to this pathogen to wheat seeds prior to planting.However, where the growth of G. gramminis is effectively under control,R. solani may become a growing problem pathogen of wheat. The geneactivating sequence for activation of genes which are effective againstR. solani could be introduced into the biocontrol strains currently usedto protect wheat from take-all to extend their range of effectiveness toinclude R. solani.

The present invention comprises an isolated DNA sequence consistingessentially of the 2 kb fragment deposited as pCIB 137, or of the ORF 5sequence shown in SEQUENCE ID No. 1. These DNA sequences are capable ofactivating latent gene activity in a bacterial strain. Thus, the presentinvention also comprises methods of activating latent gene activity in ahost bacterial strain comprising introducing the DNA sequence into thegenome of a host bacterial strain. In preferred embodiments of theinvention, the host bacterial strain may be a pseudomonad, particularlystrains of the species Pseudomonas fluorescens.

The present invention further comprises recombinant DNA sequences inwhich a bacterial regulatory element is operably linked to the DNAsequence of SEQUENCE ID No. 1. The bacterial regulatory element may be apromoter from a gene isolated from Pseudomonas, Bacillus, or E. coil. Inone embodiment of the present invention, the bacterial regulatoryelement is the native promoter of ORF 5. The bacterial regulatoryelement may be from a gene which is homologous or heterologous to thehost bacterial strain.

The present invention also includes methods of activating latent geneactivity in a host bacterial strain by transforming the host bacterialstrain with the recombinant DNA sequences of the present invention. In aparticular embodiment of the present invention, the transformed host 5bacterial strain is rendered active against fungal pathogens, such asRhizoctonia solani, Helminthosporium gramineae and species of the generaPythium and Fusarium.

The present invention further comprises isolated DNA sequences encodingthe lemA gene. These sequences are capable of restoring the productionof hydrolytic enzymes such as chitinase and gelatinase and theproduction of antifungal secondary metabolites such as pyrrolnitrin andcyanide in some mutants lacking these functions. The invention furthercomprises recombinant DNA sequences in which a bacterial regulatoryelement is operably linked to the DNA coding sequence of lemA. Thebacterial regulatory element may be a promoter from a gene isolated fromPseudomonas, Bacillus, or E. coil. In one embodiment of the presentinvention, the bacterial regulatory element is the native promoter oflemA. The bacterial regulatory element may also be from a gene which ishomologous or heterologous to the host bacterial strain.

The present invention further comprises isolated DNA sequences encodingthe rpoS gene. These sequences are capable of restoring the productionof hydrolytic enzymes such as chitinase and gelatinase and theproduction of antifungal secondary metabolites such as pyrrolnitrin andcyanide in some mutants lacking these functions. The invention furthercomprises recombinant DNA sequences in which a bacterial regulatoryelement is operably linked to the DNA coding sequence of rpoS. Thebacterial regulatory element may be a promoter from a gene isolated fromPseudomonas, Bacillus, or E. coli. In one embodiment of the presentinvention, the bacterial regulatory element is the native promoter ofrpoS. The bacterial regulatory element may also be from a gene which ishomologous or heterologous to the host bacterial strain.

The present invention also includes methods of activating genes whoseexpression is regulated by Gene Activating sequences by introducing therecombinant Gene Activating sequences of the present invention, alone orin combination, into the host bacterial strain. Preferred for use asGene Activating Sequences in this method are the gafA, lemA and rpoSgenes, and any combination thereof. In a particular embodiment of thepresent invention, the transformed host bacterial strain is renderedactive against fungal pathogens, such as Rhizoctonia solani,Helminthosporium gramineae and species of the genera Pythium andFusarium.

The present invention also includes a method for the isolation ofbiosynthetic genes for anti-pathogenic substances which utilizes theGene Activating Sequences which control their expression.

Examples of the gene activating sequences of the present invention havebeen deposited. Accordingly, the gene activating sequence includes thedeposited DNA sequence as well as fragments thereof By fragments isintended a DNA sequence which is capable of functioning as a geneactivating sequence.

DEFINITIONS

As used in the present application, the following terms have the meaningset out below:

Antipathogenic Substance (APS): A substance which requires one or morenonendogenous enzymatic activities foreign to a plant to be produced ina host where it does not naturally occur, which substance has adeleterious effect on the multiplication or growth of a pathogen (i.e.pathogen). By "nonendogenous enzymatic activities" is meant enzymaticactivities that do not naturally occur in the host where theantipathogenic substance does not naturally occur. A pathogen may be afungus, bacteria, nematode, virus, viroid, insect or combinationthereof, and may be the direct or indirect causal agent of disease inthe host organism. An antipathogenic substance can prevent themultiplication or growth of a phytopathogen or can kill a phytopathogen.An antipathogenic substance may be synthesized from a substrate whichnaturally occurs in the host. Alternatively, an antipathogenic substancemay be synthesized from a substrate that is provided to the host alongwith the necessary nonendogenous enzymatic activities. An antipathogenicsubstance may be a carbohydrate containing antibiotic, a peptideantibiotic, a heterocyclic antibiotic containing nitrogen, aheterocyclic antibiotic containing oxygen, a heterocyclic antibioticcontaining nitrogen and oxygen, a polyketide, a macrocyclic lactone, anda quinone. Antipathogenic substance is abbreviated as "APS" throughoutthe text of this application.

Promoter or Regulator DNA sequence: An untranslated DNA sequence whichassists in, enhances, or otherwise affects the transcription,translation or expression of an associated structural DNA sequence whichcodes for a protein or other DNA product. The promoter DNA sequence isusually located at the 5' end of a translated DNA sequence, typicallybetween 20 and 100 nucleotides to the 5' end of the starting site fortranslation.

Structural or Coding DNA sequence: A DNA sequence that is translated ortranscribed in an organism to produce an RNA, a protein or other DNAproduct.

Associated with/operably linked: Two DNA sequences which are"associated" or "operably linked" are related physically orfunctionally. For example, a promoter or regulator DNA sequence is saidto be "associated with" a DNA sequence that codes for an RNA or aprotein if the two sequences are operably linked, or situated such thatthe regulator DNA sequence will affect the expression level of thecoding or structural DNA sequence.

Derived from: A first DNA sequence or fragment is said to be "derivedfrom" a second DNA sequence or fragment if the former is physicallyisolated from the latter, or if the former is isolated based on sequencehomology by using pan or all of the latter as a probe for isolation. Thedegree of sequence homology is sufficient to allow specifichybridisation of the probe to the first DNA sequence and is 50% orgreater, preferably greater than 60% and most preferably greater than75%.

Homologous: A DNA sequence is said to be "homologous" to a hostorganism, such as a bacterial strain, if that DNA sequence wasoriginally isolated from, or naturally originates in, the genome of anorganism of similar biological classification as the host organism. Forexample, where a host organism to be transformed is of the speciesPseudomonas fluorescens, a DNA sequence is homologous if it originatesfrom a pseudomonad strain, particularly from a strain of the genusPseudomonas, especially the species Pseudomonas fluorescens. The term"heterologous" is used to indicate a recombinant DNA sequence in whichthe promoter or regulator DNA sequence and the associated DNA sequenceare isolated from organisms of different biological classification.

Chimeric construct/chimeric DNA sequence: A recombinant DNA sequence inwhich a regulator or promoter DNA sequence is associated with, oroperably linked to, a DNA sequence that codes for an mRNA or which isexpressed as a protein, such that the regulator DNA sequence is able toregulate transcription or expression of the associated DNA sequence. Theregulator DNA sequence of the chimeric construct is not normallyoperably linked to the associated DNA sequence as found in nature.

Genome: The term "genome" refers to the entire native genetic content ofan organism. The genome of bacterial organisms may include both thechromosomal and plasmid DNA content of an organism.

Gene activating sequences: Sequences which, when transformed into ahost, have the ability to turn on other genes which are not expressed(i.e. latent) or expressed at low levels in the naturally occurringstate of the host. These sequences typically encode proteins which playa role in the pathways which regulate gene expression. These sequencesinclude, but are not limited to, the gafA, lemA and rpoS genes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. This figure shows restriction maps of three cosmid clones,pANT5, pANT9 and pANT10, that were found to complement the ANT⁻phenotype of mutant 2-1. In this figure, `B` indicates a BamHIrestriction site; `E` an EcoRI restriction site; and `H` a HindIIIrestriction site.

FIG. 2. This figure shows the ability of DNA subfragments derived fromthe pANT5 clone of Pseudomonas fluorescens strain 915 to complement theANT⁻ phenotype of mutant 2-1 and the wild type strains 914 and 922. Thesubfragment labeled 3 is the approximately 11 kb region which has beencalled the E11 fragment.

FIG. 3. This figure indicates the organization of the E11 fragment. Thefigure indicates the location of five identified open reading frames(ORF) and restriction sites for various enzymes.

FIG. 4. This figure shows the ability of DNA subfragments derived fromthe clone pCIB 146 of Pseudomonas fluorescens strain 915 to complementthe mutant CGP 21.

DETAILED DESCRIPTION OF THE INVENTION

Pseudomonas fluorescens strain 915 was isolated from the roots of acotton plant grown in a Texas cotton field and was identified as aneffective biocontrol strain of Pythium ultimum- and Rhizoctoniasolani-induced damping off of cotton. We have determined that certainmutant derivatives of the bacterial biological control strainPseudomonas fluorescens strain 915 are deficient or altered in a varietyof functions. Such pleiotropic mutants can be isolated followingmutagenesis techniques known to those skilled in the art (e.g.nitrosoguanidine mutagenesis, transposon mutagenesis) or can arisespontaneously. One such mutant, obtain after mutagenesis with thechemical mutagen nitrosoguanidine was designated mutant 2-1. Sevenfurther mutants were identified by introducing the transposon TnCIB116into strain 915. These mutants can be identified on the basis of theirinability to inhibit in vitro the growth of the phytopathogenic fungusRhizoctonia solani. They also fail to synthesize the antifungalmetabolite pyrrolnitrin, and no longer produce cyanide or the enzymechitinase, each of which has the potential to inhibit fungal growth(Voisard et al., EMBO J. 8:351-358 (1989); Jones et al., EMBO J.5:467-473 (1986)). The mutants' production of an enzyme with gelatinaseactivity is significantly reduced, and they have an altered colonymorphology. A summary comparing the characteristics of the pleiotropicmutants with the corresponding characteristics of wild-type P.fluorescens strain 915 is presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Characteristic                                                                             P. fluorescens strain 915                                                                    Pleiotropic mutants                               ______________________________________                                        Pyrrolnitrin +              -                                                 Cyanide      +              -                                                 Chitinase    +              -                                                 Gelatinase   +              reduced                                           Colony morphology                                                                          circular, entire,                                                                            circular, undulate,                                            convex and opaque                                                                            flat and translucent                              Inhibition of Rhizoctonia                                                                  +              -                                                 solani                                                                        ______________________________________                                    

A total of eight pleiotropic mutants was identified. These all have thephenotype described in Table 1 above and fall into two distinct geneticclasses, those which can be restored to 915-phenotype by introduction ofthe gafA gene (see A below) and those which can be restored to915-phenotype by the introduction of the lemA gene (see B below).

A. Mutant Complementation with the gafA gene

An 11 kilobase EcoRl restriction fragment (referred to as fragment"E11") of P. fluorescens strain 915 was identified on the basis of itsability to restore antibiosis to a mutant, designated strain 2-1, andtwo further mutants (derived from insertion mutagenesis) which wereotherwise incapable of inhibiting the growth of the phytopathogenRhizoctonia solani in vitro or in greenhouse biological control assays.The 11 kilobase EcoRI restriction fragment (fragment E11 ) of P.fluorescens strain 915, and a 2.0 kb XhoI subclone of this fragmentcontaining gafA (ORF 5), each restored all of the lost or alteredfunctions listed in Table 1 when introduced by conjugation into oneclass of pleiotropic mutants derived from strain 915. Introduction offragment E11 or the 2.0 kb XhoI subclone into the P. fluorescens strains914 and 922 unexpectedly activate the expression of latent genesinvolved in the synthesis of pyrrolnitrin, cyanide, and chitinase, andin the case of P. fluorescens strain 914, cause an alteration in colonymorphology on minimal medium from large, circular, flat, translucent,with undulate edge to small, circular, convex, opaque white, with entireedge. Accompanying these phenotypic changes associated with theintroduction of fragment E11 or the 2.0 kb XhoI subclone is theconversion of P. fluorescens strains 914 and 922 to effective biocontrolstrains with activity against the phytopathogen Rhizoctonia solani. Wehave also demonstrated that introduction of an Escherichia coil genehomologous to ORF 5 (gafA) into P. fluorescens strain 914 activates theexpression of genes involved in the synthesis of cyanide, chitinase, andpyrrolnitrin. This result indicates that genes of the gafA class aresufficient for the activation of latent genes in heterologous bacterialstrains.

DNA sequence analysis of fragment E11 has, to date, allowedidentification of five open reading flames, as well as a tRNA gene(glyW). The organization of these open reading frames within fragmentE11 is depicted in FIG. 3. The first potential gene regulation elementwe identified is ORF 2, which shared homology with numerous sensorcomponents of bacterial two-component regulatory systems (reviewed inAlbright et al., Annu. Rev. Genet. 23:311-336 (1989)). We determinedthat the organization of glyW, ORF 3 (which has homology to theEscherichia coli gene pgsA) and ORF 4 (which has homology to the E. coligene uvrC) is identical to the gene organization found near map position42 of the E. coli genome. In E. coli, at a position equivalent to ORF 5(gafA), exists a putative transcriptional activator gene of unknownfunction (Moolenaar et al., Nucl. Acids Res. 15:4273-4289 (1987)). ORF 5(gafA) exhibits homology to this putative activator gene. Furthermore,comparison of the fragment E11 sequence with DNA sequences contained inthe Genbank database reveals that ORF 5 has substantial homology to aproposed transcriptional activator gene isolated from Pseudomonasfluorescens CHA0 by Laville et al., Proc. Natl. Acad. Sci. USA89:1562-1566 (1992). Thus, two of the open reading frames, ORF 2 and ORF5, share significant homology with numerous sensor and activatorcomponents, respectively, of bacterial two-component regulatory systems(reviewed in Albright et al., Annu. Rev. Genet. 23:311-336 (1989)).

Subcloning experiments are performed with fragment E11 with the aim ofdetermining whether the gene(s) responsible for restoring lost functionsto the pleiotropic mutants and for activating latent activities inheterologous Pseudomonas strains could be isolated on a smallerrestriction fragment. A 2.0 kb XhoI subclone containing ORF 5 isprepared, as is a 3.7 kb EcoRI-XbaI subclone containing ORF1 and ORF 2.The 2.0 kb XhoI subclone is sufficient to restore the lost functions inthe class of pleiotropic mutants originally complemented by fragment E11and activated the expression of latent genes in P. fluorescens strains914 and 922. The 3.7 kb EcoRI-XbaI subclone had no measurable effect.Furthermore, when the gala gene was cloned from the strain 914,transferred to plasmid pLAFR3 and reintroduced into strain 914 thelatent genes are activated indicating that strain 914 does contain agafA gene capable of functioning, but that the expression of the galagene in strain 914 is presumably not at levels high enough to activatethe latent genes.

To determine whether transcriptional activators of the gafA class aregenerally capable of activating the expression of latent genes inheterologous bacterial strains, we cloned the putative transcriptionalactivator gene described by Moolenar et al. and introduced it into P.fluorescens strain 914. The E. coli gene, which encodes a protein whichis approximately 60% homologous to that encoded by gafA, activated theexpression of genes involved in the production of cyanide, chitinase,and pyrrolnitrin in P. fluorescens strain 914.

It is an aspect of the present invention that improved biologicalcontrol strains can be identified following the introduction oftranscriptional activators of the gafA class into a variety ofenvironmental isolates. This approach represents a method for theidentification of potentially effective biocontrol strains which wouldotherwise not be selected by any of the screening methods currentlyavailable.

Those skilled in the an will also be aware that it will be possible toimprove a biological control strain by placing additional genes underthe control of transcriptional activators of the gafA class. This can beaccomplished by identifying the gafA-responsive promoter element(s) andoperably linking the desired gene or genes to such element(s) beforeintroducing such genes into the desired strain.

In one embodiment of the present invention, recombinant DNA sequencesare obtained which comprise an approximately 2 Kb DNA sequenceconsisting essentially of the DNA sequence of gafA. This DNA sequencedemonstrates pleiotropic effects of activating latent gene activity orincreasing the efficacy of other genes. Among the pleiotropic effects ofthe gafA DNA sequence are the increased ability to inhibit the growth offungal pathogens, such as Rhizoctonia solani, Helminthosporium gramineaeand species of the genera Pythium and Fusarium This DNA sequence may bederived from bacterial strains which are effective biocontrol agentsagainst Rhizoctonia. Preferably the DNA sequence may be derived from theclone pANT5, which was isolated from a strain of Pseudomonasfluorescens. More preferably, the DNA sequence may comprise theapproximately 11 kb E11 fragment of pANT5. In particular embodiments ofthe invention, the DNA sequence consists essentially of theapproximately 2 kb fragment, or the DNA sequence of SEQUENCE ID No. 1.The clone pANT5 has been deposited with ATCC and has been designatedATCC accession number 40868. A plasmid containing the 11 kb E11 fragmentof pANT5 has been deposited with ATCC and has been designated ATCCaccession number 40869. The approximately 2 kb ORF 5 DNA sequence may beobtained from the E11 fragment of pANT5 as a 2 kb fragment afterdigestion with XhoI. This fragment has been designated pCIB137 and hasbeen deposited with the USDA Agricultural Research Service CultureCollection, Northern Regional Research Center (NRRL).

The recombinant DNA sequences of the present invention may be chimericand may be heterologous or homologous. The recombinant DNA sequences ofthe present invention may further comprise one or more regulatory DNAsequences operably linked to the structural DNA sequence above. Suchregulatory DNA sequences include promoter sequences, leader sequences,and other DNA sequences which may affect the expression of theregulatory DNA sequences, as well as those fragments of a regulator DNAsequence that are able to act with such effect.

B: Mutant Complementation with the lemA Gene

Of the eight pleiotropic mutants isolated, five are not complemented byplasmids carrying the gafA gene indicating that at least one othergenetic locus is required for gene activation. A total gene library ofstrain 915 was introduced into these mutants (e.g. CGP 21) byconjugation and transconjugants which had regained wildtype morphologyare obtained. These transconjugants also produced pyrrolnitrin,chitinase, and cyanide. The restoring clones are isolated from thetransconjugants and characterized. A 6 kb subclone which encodes a genewith high homology to lemA (Hrabak et al., (1992) supra) was found toretain the phenotype restoration ability. This clone was deposited aspCIB 168. Consequently the lemA gene clearly has the ability to restorethe biocontrol phenotype in these pleiotropic mutants. However, when thelemA gene was introduced into strain 914 it was not capable ofactivating latent gene expression, corroborating the assertion thatstrain 914 does not produce chitinase, gelatinase, pyrrolnitrin andcyanide because of inadequate gafA expression. By way of corollary, lemAwould predictably activate latent gene expression in strains ofPseudomonas in which lemA expression is the rate-limiting factorpreventing the production of chitinase, gelatinase, pyrrolnitrin andcyanide. Other bacterial genes functionally homologous to lemA would beable to act in the same way as lemA. Such genes comprise a class ofsensory component genes capable of phosphorylating and thereforeactivating the gafA class of activators described above. Members of theclass can be identified and isolated by complementation of theappropriate class of pleiotropic mutants as described herein. Forexample, the corresponding E. coli gene responsible for thephosphorylation of uvrY is one such gene.

Clearly the gene activating sequences gafA and lemA play pivotal rolesin the activation of a series of genes involved in the production ofenzymes and metabolites important in the biocontrol phenotype ofPseudomonas.

The rpoS gene: An Additional Gene Activating Sequence

In Escherichia coli, it has been demonstrated that an alternate sigmafactor designated rpoS is required for the transcription of a large setof genes which are preferentially expressed during stationary phaseconditions (i.e. conditions of nutrient stress) (Siegele and Kolter, J.Bacteriol. 174:345-348 (1992); Hengge-Aronis, Cell 72:165-168 (1993)).The homologue of the E. coli rpoS gene was isolated from the P.fluorescens strain 915. The DNA sequence of the P. fluorescens strain915 rpoS gene and the deduced amino acid sequence of the rpoS sigmafactor are provided as Sequence ID No. 8 and Sequence ID No. 9,respectively. The deduced amino acid sequence is greater than 86%identical to that of the unpublished sequence of a Pseudomonasaeruginosa rpoS gene (Tanaka and Takahashi, GenBank accession #D26 134,(1994)). It is also highly homologous to the E. coli, Salmonellatyphimurium, and Shigella flexneri RpoS sigma factors (Mulvey andLoewen, Nucleic Acids Res. 17:9979-9991 (1989), GenBank accession#U05011, GenBank accession #U00119).

According to the present invention the rpoS gene may be used tostimulate biocontrol factors and secondary metabolites. The expressionpattern of rpoS can be altered by placing it behind bacterial regulatoryelements. Altering the temporal expression of rpoS, which is itselfnormally induced at the onset of stationary phase (Tanaka et al., Proc.Natl. Acad. Sci. 90:3511-3515 (1993)) has the effect of inducingexpression of rpoS regulated genes earlier than normal, leading toincreased production of biocontrol factors and other secondarymetabolites with biological activity. This results, for example inenhanced production of pyrrolnitrin, chitinase, cyanide, gelatinase,etc. and increases the biocontrol efficacy of P. fluorescens strain 915and heterologous strains with rpoS-regulated biocontrol factors. TherpoS gene with altered expression may be cloned into broad-host-rangeplasmids, shuttle vectors, etc. for introduction by conjugation,transformation, electroporation, transduction, etc. into a wide varietyof bacteria for the stimulation of secondary metabolites, exportedenzymes, etc. as a screening strategy for new biocontrol agents andnovel metabolites with biological activity (antifungal, insecticidal,herbicidal, anti-bacterial, etc).

It is a further aspect of the present invention that improved biologicalcontrol strains can be identified following the introduction oftranscriptional activators of the rpoS class into a variety ofenvironmental isolates. This approach represents a method for theidentification of potentially effective biocontrol strains which wouldotherwise not be selected by any of the screening methods currentlyavailable.

Those skilled in the art will also be aware that it will be possible toimprove a biological control strain by placing additional genes underthe control of transcriptional activators of the rpoS class. This can beaccomplished by identifying the rpoS-responsive promoter element(s) andoperably linking the desired gene or genes to such element(s) beforeintroducing such genes into the desired strain.

The Use of Gene Activating Sequences for Plant Pathogen Control

In another embodiment of the present invention, biocontrol agents areprovided which are able to inhibit the growth of fungal pathogens, suchas Rhizoctonia solani, Helminthosporium gramineae and species of thegenera Pythium and Fusarium. These biocontrol agents may be bacteria,plant cells or animal cells transformed with the recombinant DNAsequences above, but are preferably bacterial strains, and morepreferably gram negative bacterial strains, such as the pseudomonads.Most preferred as the biocontrol agent are strains of the speciesPseudomonas fluorescens.

Another embodiment of the present invention provides methods ofinhibiting the growth of fungal pathogens, such as Rhizoctonia solani,Helminthosporium gramineae and species of the genera Pythium andFusarium. In the methods of the present invention, the gene activatingDNA sequences can be introduced into the genome of a bacterial strainwhich may not ordinarily be effective as an inhibitor of fungalpathogens, resulting in an effective biocontrol strain.

DNA in the form of plasmids can be transferred from one bacterium toanother by a sexual process termed conjugation. Plasmids capable ofconjugal transfer contain genes that code for the synthesis of sex pili.Sex pili are hollow tubes that join the plasmid,containing bacterium(the donor) with another bacterium (the recipient) and through whichreplicated copies of the plasmid pass from the donor to the recipient.This procedure occurs naturally in nature and is utilized in thelaboratory as a method of transferring genes from one bacterium toanother. For some strains of bacteria, such as Pseudornonas, conjugaltransfer of DNA is the preferred method since these bacteria are notreadily transformed with extraneous DNA.

In yet another embodiment of the present invention, methods are providedfor producing antibiotic substances which are effective in inhibitingthe growth of fungal pathogens, such as Rhizoctonia solani,Helminthosporium gramineae and species of the genera Pythium andFusarium. This method comprises introducing the recombinant DNAsequences of the present invention into the genome of a biocontrol agentto form a transformed biocontrol agent, allowing the transformedbiocontrol agent to produce antibiotic substances, such as pyrrolnitrin,and extracting the antibiotic substance from the transformed biocontrolagent.

The present invention embraces the preparation of antifungalcompositions in which one or more of the transformed biocontrolbacterial strains are used as active ingredient. The present inventionfurther embraces the preparation of antifungal compositions in which theactive ingredient is the antifungal metabolite or antibiotic compoundproduced by the transformed biocontrol agent of the present invention.Where the active ingredient is a biocontrol bacterial strain, thebiocontrol preparation may be applied in any manner known for seed andsoil treatment with bacterial strains. The bacterial strain may behomogeneously mixed with one or more compounds or groups of compoundsdescribed herein, provided such compound is compatible with bacterialstrains. The present invention also relates to methods of treatingplants, which comprise application of the bacterial strain, orantifungal compositions containing the bacterial strain, to plants.

The active ingredient of the present invention may also be an antifungalmetabolite, such as an antibiotic compound, produced by the biocontrolagents of the present invention. The present invention also relates tomethods of treating plants, which comprise application of the antifungalmetabolite, such as an antibiotic compound, or antifungal compositionscontaining the metabolite, to plants.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with further compounds.These compounds can be both fertilizers or micronutrient donors or otherpreparations that influence plant growth. They can also be selectiveherbicides, insecticides, fungicides, bactericides, nematicides,mollusicides or mixtures of several of these preparations, if desiredtogether with further carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers.

A preferred method of applying active ingredients of the presentinvention or an agrochemical composition which contains at least one ofthe active ingredients is leaf application. The number of applicationsand the rate of application depend on the intensity of infestation bythe corresponding pathogen (type of fungus). However, the activeingredients can also penetrate the plant through the roots via the soil(systemic action) by impregnating the locus of the plant with a liquidcomposition, or by applying the compounds in solid form to the soil,e.g. in granular form (soil application). The active ingredients mayalso be applied to seeds (coating) by impregnating the seeds either witha liquid formulation containing active ingredients, or coating them witha solid formulation. In special cases, further types of application arealso possible, for example, selective treatment of the plant stems orbuds.

The active ingredients are used in unmodified form or, preferably,together with the adjuvants conventionally employed in the an offormulation, and are therefore formulated in known manner toemulsifiable concentrates, coatable pastes, directly sprayable ordilutable solutions, dilute emulsions, wettable powders, solublepowders, dusts, granulates, and also encapsulations, for example, inpolymer substances. Like the nature of the compositions, the methods ofapplication, such as spraying atomizing, dusting, scattering or pouting,are chosen in accordance with the intended objectives and the prevailingcircumstances. Advantageous rates of application are normally from 50 gto 5 kg of active ingredient (a.i.) per hectare ("ha", approximately2.471 acres), preferably from 100 g to 2 kg a.i./ha, most preferablyfrom 200 g to 500 g a.i./ha.

The formulations, compositions or preparations containing the activeingredients and, where appropriate, a solid or liquid adjuvant, areprepared in known manner, for example by homogeneously mixing and/orgrinding the active ingredients with extenders, for example solvents,solid carriers and, where appropriate, surface-active compounds(surfactants).

Suitable solvents include aromatic hydrocarbons, preferably thefractions having 8 to 12 carbon atoms, for example, xylene mixtures orsubstituted naphthalenes, phthalates such as dibutyl phthalate ordioctyl phthalate, aliphatic hydrocarbons such as cyclohexane orparaffins, alcohols and glycols and their ethers and esters, such asethanol, ethylene glycol monomethyl or monoethyl ether, ketones such ascyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone,dimethyl sulfoxide or dimethyl formamide, as well as epoxidizedvegetable oils such as epoxidized coconut oil or soybean oil; or water.

The solid carriers used e.g. for dusts and dispersible powders, arenormally natural mineral fillers such as calcite, talcum, kaolin,montmorillonite or attapulgite. In order to improve the physicalproperties it is also possible to add highly dispersed silicic acid orhighly dispersed absorbent polymers. Suitable granulated adsorptivecarriers are porous types, for example pumice, broken brick, sepioliteor bentonite; and suitable nonsorbent carriers are materials such ascalcite or sand. In addition, a great number of pregranulated materialsof inorganic or organic nature can be used, e.g. especially dolomite orpulverized plant residues.

Depending on the nature of the active ingredient to be used in theformulation, suitable surface-active compounds are nonionic, cationicand/or anionic surfactants having good emulsifying, dispersing andwetting properties. The term "surfactants" will also be understood ascomprising mixtures of surfactants.

Suitable anionic surfactants can be both water-soluble soaps andwater-soluble synthetic surface-active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts orunsubstituted or substituted ammonium salts of higher fatty acids(chains of 10 to 22 carbon atoms), for example the sodium or potassiumsalts of oleic or stearic acid, or of natural fatty acid mixtures whichcan be obtained for example from coconut oil or tallow oil. The fattyacid methyltaurin salts may also be used.

More frequently, however, so-called synthetic surfactants are used,especially fatty sulfonates, fatty sulfates, sulfonated benzimidazolederivatives or alkylarylsulfonates.

The fatty sulfonates or sulfates are usually in the form of alkali metalsalts, alkaline earth metal salts or unsubstituted or substitutedammoniums salts and have a 8 to 22 carbon alkyl radical which alsoincludes the alkyl moiety of alkyl radicals, for example, the sodium orcalcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixtureof fatty alcohol sulfates obtained from natural fatty acids. Thesecompounds also comprise the salts of sulfuric acid esters and sulfonicacids of fatty alcohol/ethylene oxide adducts. The sulfonatedbenzimidazole derivatives preferably contain 2 sulfonic acid groups andone fatty acid radical containing 8 to 22 carbon atoms. Examples ofalkylarylsulfonates are the sodium, calcium or triethanolamine salts ofdodecylbenzenesulfonic acid, dibutylnapthalenesulfonic acid, or of anaphthalenesulfonic acid/formaldehyde condensation product. Alsosuitable are corresponding phosphates, e.g. salts of the phosphoric acidester of an adduct of p-nonylphenol with 4 to 14 moles of ethyleneoxide.

Non-ionic surfactants are preferably polyglycol ether derivatives ofaliphatic or cycloaliphatic alcohols, or saturated or unsaturated fattyacids and alkylphenols, said derivatives containing 3 to 30 glycol ethergroups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moietyand 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts ofpolyethylene oxide with polypropylene glycol, ethylenediamine propyleneglycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms inthe alkyl chain, which adducts contain 20 to 250 ethylene glycol ethergroups and 10 to 100 propylene glycol ether groups. These compoundsusually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants renonylphenolpolyethoxyethanols, castor oil polyglycol ethers,polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethylene glycol andoctylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitanand polyoxyethylene sorbitan trioleate are also suitable non-ionicsurfactants.

Cationic surfactants are preferably quaternary ammonium salts whichhave, as N-substituent, at least one C₈ -C₂₂ alkyl radical and, asfurther substituents, lower unsubstituted or halogenated alkyl, benzylor lower hydroxyalkyl radicals. The salts are preferably in the form ofhalides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammoniumchloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation aredescribed, for example, in "McCutcheon's Detergents and EmulsifiersAnnual," MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely andWood, "Encyclopedia of Surface Active Agents," Chemical Publishing Co.,Inc. New York, 1980.

The agrochemical compositions usually contain from about 0.1 to about99%, preferably about 0.1 to about 95%, and most preferably from about 3to about 90% of the active ingredient, from about 1 to about 99.9%,preferably from abut 1 to about 99%, and most preferably from about 5 toabout 95% of a solid or liquid adjuvant, and from about 0 to about 25%,preferably about 0.1 to about 25%, and most preferably from about 0.1 toabout 20% of a surfactant.

Whereas commercial products are preferably formulated as concentrates,the end user will normally employ dilute formulations.

Further Uses of Gene Activating Sequences

This invention also describes a novel technique for the isolation of APSbiosynthetic genes which utilizes the Gene Activating Sequences whichcontrol their expression. In this method, a library of transposoninsertion mutants is created in a strain of microorganism which lacksthe Gene Activating Sequence or has had the Gene Activating Sequencedisabled by conventional gene disruption techniques. The insertiontransposon used carries a promoter-less reporter gene (e.g. lacZ). Oncethe insertion library has been made, a functional copy of the GeneActivating Sequence is transferred to the library of cells (e.g. byconjugation or electroporation) and the plated cells are selected forexpression of the reporter gene. Cells are assayed before and aftertransfer of the Gene Activating Sequence. Colonies which express thereporter gene only in the presence of the Gene Activating Sequence areinsertions adjacent to the promoter of genes regulated by the GeneActivating Sequence. Assuming the Gene Activating Sequence is specificin its regulation for APS-biosynthetic genes, then the genes tagged bythis procedure will be APS-biosynthetic genes. In a preferredembodiment, the cloned Gene Activating Sequence is the gafA genedescribed in PCT application WO 94/01561 which regulates the expressionof the biosynthetic genes for pyrrolnitrin. Thus, this method is apreferred method for the cloning of the biosynthetic genes forpyrrolnitrin.

It is recognized that the gene activating sequences of the presentinvention can be used in a variety of microorganisms to induce theproduction of gene products and secondary metabolites. The activatingsequences are capable of inducing or enhancing the expression of geneswhich may be latent or natively expressed at low levels. The activatingsequences may be used individually or in various combinations to induceor enhance expression of the genes they regulate. Preferred for use asgene activating sequences in such methods are the gafA, lemA and rpoSgenes and combinations thereof

As discussed above, the activating elements find use in the productionof antibiotics in microorganisms for the purposes of biocontrol. Suchelements are also useful in the production of antibiotics forpharmaceutical purposes, particularly in strains of microorganisms whichnatively express the biosynthetic genes at low levels or not at all.Examples of strains suitable for manipulation in this manner includestrains of Streptomyces for the production of tetracycline,erythromycin, chloromycetin, and streptomycin; strains of Bacillus forthe production of bacitracin; and strains of Penicillium for theproduction of penicillin.

The activating elements find further use in the production of vitamins,growth factors and hormones in selected strains of microorganisms.Examples include the enhanced production of vitamin B2 and B12 fromAshbya and Streptomyces, respectively; the production of gibberellinfrom Fusarium and Gibberella; the production of 11-α-hydroxyprogesteronefrom Rhizopus; and the production of corticosterone from Curvularia.

Other applications include but are not limited to the enhancedproduction of butanol, acetone, and ethanol from Clostridium; theproduction of glycerol from Saccharomyces; the production of lactic acidfrom Lactobacillus; the production of polysaccharides such as alginate,xanthan, dextran from Leuconostoc and other organisms; the production ofsecreted proteins and enzymes such as amalyses, proteases, pectinases,chitinases, cellulases, gelatinases, collagenases, elastases, etc. fromBacillus, Aspergillus and other organisms; and the production ofinvertase from Saccharomyces.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1

Mutant Isolation and Functional Complementation

The wild-type Pseudomonas fluorescens strain 11c-1-38 (strain 915) wasmutated by exposure to the mutagen N-Methyl-N'-nitro-N-nitrosoguanidine.Approximately two dozen antibiotic-affected mutants were identified byscreening individual mutants for their ability to inhibit the growth ofP. ultimum and R. solani on nutrient agar. Most of these were shown tohave reduced, or no, antibiotic activity against one of the twophytopathogenic fungi, but were not affected in their inhibition of theother fungus. However, one mutant, that we have named mutant 2-1, wasfound to lack antibiotic activity against both test fungi. These resultsstrongly indicated that P. fluorescens strain 11c-1-38 produced twodistinct antibiotics, one effective against P. ultimum and the othereffective against R. solani, rather than one antibiotic that waseffective against both fungi.

A gene library of total DNA isolated from the parent strain wasconstructed by partial SalI restriction of the DNA, size fractionationto yield fragments of 20-30 kilobases, and ligation into XhoI-restrictedvector pVK100. Knauf et al., Plasmid 8:45-54 (1982). The gene librarywas transferred to the antibiotic mutant 2-1 by triparental conjugationwith E. coli harboring the tra⁺ plasmid pRK2013. Ditta et al., Proc.Natl. Acad Sci. USA 77:7347-7351 (1980). Transgenic exconjugants weretested for the production of antibiotic by measuring growth inhibitionof P. ultimum and R. solani. Three overlapping clones were identifiedthat restored antibiotic activity against R. solani, but not against P.ultimum, to mutant 2-1. Restriction maps of these clones were determinedand are shown in FIG. 1.

Example 2

Characterization of the E11 gene region

The genetic region necessary for the functional complementation ofantibiotic biosynthesis in mutant 2-1 was defined by subcloning portionsof the larger region and assessing their ability to complement mutant2-1 for antibiosis (FIG. 2). The smallest fragment that was demonstratedto complement the mutation was the 4.9 kb HindIII/EcoRI fragment, whichwas indicated as subfragment 6 in FIG. 2 and was designated subfragmentH/E4.9. Some of the subcloned DNA fragments derived from the antibioticgene parent clone, which was designated pANT-5, were transferred to twowild type P. fluorescens strains, 11c-1-33 (strain 914) and 11-1-6(strain 922), that were not ordinarily able to produce antibioticagainst R. solani.

The results, shown in FIG. 2, indicated that an 11 kb EcoRI subfragment,which is indicated as subfragment 3 in FIG. 2 and was designatedsubfragment E11, imparts R. solani-active antibiosis to both strains.

Example 3

Inhibition of Rhizoctonia solani

An active antibiotic compound can be extracted from the growth medium ofthe transformed P. fluorescens strain that produces this antibiotic.This was accomplished by extraction of the medium with 80% acetonefollowed by removal of the acetone by evaporation and a secondextraction with diethyl ether. The diethyl ether was removed byevaporation and the dried extract is resuspended in a small volume ofwater. Small aliquots of the antibiotic extract applied to small sterilefilter paper discs placed on an agar plate will inhibit the growth of R.solani, indicating the presence of the active antibiotic compound. Theantibiotic was determined by NMR and mass spectrometry to bepyrrolnitrin.

Example 4

Description of the pleiotropic defects restored by fragment E11 and bythe 2. 0 kb XhoI subfragment; ability of these fragments to activatelatent genes in other bacterial strains

The mutant derivative of P. fluorescens strain 915 designated mutant 2-1was initially isolated on the basis of its reduced ability to inhibitthe growth of the fungus Rhizoctonia solani in vitro. The production ofan antibiotic metabolite, subsequently identified as pyrrolnitrin, waslacking in mutant 2-1. While lack of pyrrolnitrin production was thefirst defect observed in mutant 2-1, additional experimentation, detailsof which are described below, reveal that mutant 2-1 is a member of oneclass of pleiotropic mutants with characteristics summarized in Table 1.

a) Loss of pyrrolnitrin production

P. fluorescens strain 915 and mutant 2-1 were tested for pyrrolnitrinproduction by growing the respective cultures for three days in 50 ml ofnutrient broth containing AMBERLITE XAD4 resin (Rohm and Haas) (5% v/v)at 28 C. The resin was collected in a sieve and washed extensively withwater. The pyrrolnitrin was eluted from the resin by two consecutiveextractions with isopropanol (0.5× volume). The two extractions werecombined and desiccated under vacuum in a rotary evaporator at 40 C. Thedesiccated material is dissolved in 2 ml of isopropanol and was furtheranalyzed by HPLC chromatography using a Hypersil ODS column (2.1 mmdia.×10 cm) with a mobile phase consisting of a water/methanol mixturewith a starting composition of 0/100% and gradually changing to a finalcomposition of 100/0%. Prior to injection into the HPLC, 100 ul of eachextract was desiccated under vacuum and resuspended in the same volumeof water/methanol (50/50). The material eluting from the column wasmonitored by B absorbance at 212 nm and at 252 nm, and was fractionatedby elution time. P. fluorescens strain 915 extracts contained a peakwhich comigrates with a pyrrolnitrin standard and which was determinedto be pyrrolnitrin by NMR spectroscopy and by mass spectrometry. Mutant2-1 and the other identified pleiotropic mutants lack this peak.

b.) Loss of cyanide production

P. fluorescens strain 915 and mutant 2-1 and additional pleiotropicmutants were tested for the production of cyanide. Pieces of Whatmanpaper were impregnated with 5 mg/ml chloroform cupric ethyl acetoacetateand 5 mg/ml chloroform 4,4'-methylene bis-(N,N dimethyl aniline) andchloroform was allowed to evaporate. Papers were then placed under thecovers of microtiter plates whose wells have been inoculated withcultures of strain 915 and the various pleiotropic mutants. Plates arewrapped in aluminum foil and incubated overnight at 28 C. The paperturned a blue color above the well of each culture producing cyanide.The results indicated that strain 915 produced cyanide, while thepleiotropic mutants failed to produce cyanide.

c.) Loss of chitinase production

P. fluorescens strain 915 and various pleiotropic mutants were testedfor the presence of chitinase activity by either of two methods. In thefirst method, 300 ml L-broth cultures of each strain were incubated at28 C. for 12 hours. Cells were collected by centrifugation and washedonce in 20 mM phosphate buffer (10 mM Na2HPO4/10 mM KH2PO4). Followingcentrifugation, the cell pellets were resuspended in 5 ml of the 20 mMphosphate buffer. Cells were lysed by sonication for 60 seconds with themicrotip of a Branson sonifier and cell debris is removed from the cellextracts by centrifugation. Chitinase activity was assayed by incubationof 100 ul of cell extract with 100 ul of tritiated chitin (0.5% w/v;approximately 0.1 mCi/ml) in a 250 ul total volume of 0.03M sodiumphosphate, pH 6.5 for 1 hour at 37 C. The reaction was stopped byaddition of an equal volume of 2M TCA, followed by centrifugation. 200ul aliquots were counted in a liquid scintillation counter to determinesoluble counts released from the insoluble chitin molecules as a resultof chitinase activity. Typical results, presented in Table 2, indicatethat a transposon mutant designated #736, which proved to be a member ofone class of pleiotropic mutants, lacks the chitinase activity found inP. fluorescens strain 915.

                  TABLE 2                                                         ______________________________________                                        STRAIN          COUNTS/MINUTE                                                 ______________________________________                                        P. fluorescens 915                                                                            32205                                                         Transposon mutant #736                                                                        4366                                                          No extract      4993                                                          ______________________________________                                    

In the second method, 200 ul cultures of each strain were grownovernight at 28 C. in L-broth in the wells of a 96-well microtiterplate. The overnight cultures were frozen and allowed to thaw beforefurther use to release some enzyme which might otherwise be cell-bound.10 ul of 0.5 mM 4-methylumbelliferyl beta-D-N,N'-diacetylchitobiosidewas added to the wells of an opaque black microtiter plate containing 10ul of each overnight bacterial culture and 80 ul of a solutionconsisting of 50 mM sodium phosphate, pH 7.0; 10 mM EDTA, 0.1% TritonX-100, 0.1% Sarkosyl, and 10 mM beta-mercaptoethanol. Incubation was for3 hours at 37 C. Release of fluorescent methylumbelliferyl groups bychitinase activity was monitored by reading the microtiter plates at anexcitation of 355 nm and an emission of 460 ran on a Titertek FluoroscanII fluorescent spectrophotometer. Typical results, presented in Table 3,indicate that pleiotropic mutants lack the chitinase activity found inP. fluorescens strain 915.

                  TABLE 3                                                         ______________________________________                                        STRAIN         FLUORESCENCE UNITS                                             ______________________________________                                        P. fluorescens 915                                                                           13.2                                                           Pleiotropic mutant                                                                           0.687                                                          ______________________________________                                    

d.) Reduction in gelatinase activity

Gelatinase activity of P. fluorescens strain 915 and various pleiotropicmutant derivatives was assayed by incubating the bacteria on nutrientagar plates supplemented with 3% w/v Bacto-gelatin (Difco). A cloudyhalo forms in the agar surrounding colonies synthesizing and exporting aprotease capable of hydrolyzing the gelatin. Prominent halos appearedaround colonies of strain 915 following 24 hour incubation at 28 C. Suchhalos failed to appear around colonies of the pleiotropic mutants withinthe 24 hour time period, appearing instead after approximately 48 hours.Thus, while the pleiotropic mutants are not totally devoid of gelatinaseactivity, they either fail to synthesize the species of protease whichappears in strain 915 within 24 hours, or else the synthesis and/orexport of that species is delayed.

e.) Alteration in colony morphology

On minimal growth medium, P. fluorescens strain 915 forms small,circular, convex white opaque colonies with entire edges. Allpleiotropic mutants examined thus far formed larger, circular, flat,translucent colonies with undulate edges.

Example 5

Analysis of the 11 kilobase fragment (E11)

FIG. 3 depicts the genetic organization of the 11 kilobase fragment E11determined to date from DNA sequence analysis. A variety of subclones offragment E11 were prepared as double-stranded templates for dideoxysequencing reactions by digestion of fragment E11 with variousrestriction endonucleases, either singly or in combination, followed byligation with appropriate cloning vectors such as pBS SK+ (Stratagene).As regions of contiguous DNA sequence were generated, they are comparedagainst sequences contained in the GenBank database for homology withknown bacterial gene coding regions. This analysis has thus far led tothe assignment of the genetic organization for fragment E11 depicted inFIG. 3. ORF 1 shares substantial homology with the cheR gene of E. coliand the frzF gene of Myxococcus xanthus. cheR in E. coli has a methyltransferase activity which is involved in mediating the chermotaxisresponse (Springer and Koshland, Proc. Natl. Acad. Sci. USA 74:533-537)and frzF appears to have a similar function in M. xanthus (McCleary etal., J. Bacteriol. 172:4877-4887 (1990)). ORF 2 shares substantialhomology with numerous genes encoding sensor components of so-calledbacterial two-component regulatory sequences (Albright et al., 1989;Bourret et al., 1991; and Mekalanos, 1992). Examples of other bacterialsensor component genes include cheA of E. coli, rcsC of E. coli, frzE ofMyxococcus xanthus, and bvgS of Bordetella pertussis. The P. fluorescensstrain 915 glyW tRNA gene is 100% homologous to the glyW tRNA locus ofE. coli. ORF 3 shares substantial homology with the pgsA gene of E. coliwhich encodes phosphatidyl glycerophosphate, an enzyme involved inphospholipid metabolism (Gopalakrishnan et al., J. Biol. Chem.261:1329-1338 (1986)). ORF 4 shares substantial homology with the uvrCgene orE. coli which encodes a component of the ultraviolet light damagerepair excinuclease (Sharma et al., Nucl. Acids Res. 14:2301-2318(1986)).

ORF 5, which is present on the gene-activating 2.0 kb XhoI subclone offragment E11, shares substantial homology with numerous genes encodingtranscription activator components of bacterial two-component regulatorysequences. In particular, ORF 5 is highly similar to the gacA gene ofPseudomonas fluorescens CHA0 (Laville et al., 1992) and to the uvr-23gene of E. coli (Moolenar et al., 1987). Examples of some otherbacterial transcriptional activator genes with sequence similarity toORF 5 include sacU of Bacillus subtills, bvgA of Bordetella pertussis,and algR of Pseudomonas aeruginosa. The entire coding region of ORF 5 ispresented as SEQUENCE ID NO. 1. The entire sequence of a. 5.6 kilobaseportion of fragment E11, bounded by the left-most EcoRI site depicted inFIG. 3 and by an internal HindIII site, is presented as SEQUE ID 2. Thecoordinates of open reading flames contained in sequence ID 2 are asfollows:

ORF 1: 210-1688; transcribed left to right

ORF 2: 1906-3633; transcribed left to right

glyW: 4616-4691; transcribed right to left

ORF 3: 4731-5318; transcribed right to left

The combined results that the 2.0 kb XhoI subclone of fragment E11containing ORF 5 activates latent gene expression in Pseudomonas strainsand that the E. coli uvr-23 gene, which is homologous to ORF 5, iscapable of activating latent Pseudomonas gene expression indicate thattranscriptional activators of the ORF 5 class have the unexpectedcapability of activating the expression of latent bacterial genes.

Example 6

Cloning of 2 kb fragment

The mutant strain 2-1 was derived from the biocontrol Pseudomonas strain915 following N-Methyl-N'-nitro-N- nitrosoguanidine treatment. It isinitially identified on the basis of its inability to inhibit the growthof the fungi Rhizoctonia solani and Pythium ultimum in vitro. Furthercharacterization revealed that the strain was also defective in theexpression of a number of activities, including pyrrolnitrin, chitinase,and cyanide production. In addition, mutant 2-1 is morphologicallydistinguishable from strain 915. On agar plates containing the definedmedium LMG (0.1% KH₂ PO₄, 0.1% Na₂ HPO₄, 0.1% NaCl, 0.4% (NH₄)₂ SO₄,0.02% glucose and 0.66% MgSO₄, and 1.6% agar) the wild type parent(strain 915) formed small, circular, convex, white opaque colonies withentire edges. Mutant 2-1 formed larger, circular, flat, translucentcolonies with undulate edges.

The approximately 2 kb XhoI fragment containing ORF5 was cloned into thebroad host range plasmid pVK100 (Knauf et al., (1982), Plasmid 8:45-54)to yield the plasmid pCIB137.

Example 7

Analysis of the 2 kilobase XhoI fragment

Comparison of the ORF5 sequence to DNA sequences in the GenBank databaserevealed substantial homology with numerous transcription activatorgenes in known bacterial two-component regulatory systems. Inparticular, ORF5 is highly similar to the gacA gene of Pseudomonasfluorescens CHA0 and to the uvr-23 gene of E. coli. ORF5 is locatedentirely within an approximately 2 kb region defined by XhoI restrictionsites. This approximately 2 kb XhoI fragment was cloned into the broadhost range plasmid pVK100 (Knauf et al. (1982), Plasmid 8:45-54) toyield the plasmid pCIB137. pCIB137 was deposited with the USDAAgricultural Research Service Culture Collection, Northern RegionalResearch Center (NRRL) at 1815 North University Street, Peoria, Ill.61604 on Jun. 24, 1992 and has been accorded the accession numberB-18981.

pCIB137 was introduced into the E. coli host strain S17-1 (Simon et al.(1983), Biotechnology 1:784-791) by transformation. It was thentransferred into strain 2-1 by conjugation. Fresh overnight cultures ofS 17-1(pCIB 137) and strain 2-1 were mixed (50 μl each) on an L agarplate and allowed to incubate overnight at 28 C. Loopfuls of bacteriafrom the mating mixture were then streaked on LMG agar containing 15μg/ml tetracycline and further incubated at 28 C. Tetracycline resistantcolonies were purified and examined for the presence of pCIB137.Transconjugants of strain 2-1 containing pCIB137 were shown to producepyrrolnitrin, chitinase, and cyanide. They also display the morphologyof strain 915.

The natural Pseudomonas isolates strain 914 and strain 922 do notproduce detectable levels of pyrrolnitrin, chitinase, or cyanide. Theyformed large, circular, flat, translucent colonies with undulate edgeson LMG agar. pCIB137 is introduced into these strains by conjugation asdescribed in example 2. Transconjugants of strain 914 containing pCIB137were shown to produce pyrrolnitrin, chitinase, and cyanide. They alsodisplayed the morphology of P. fluorescens strain 915. Transconjugantsof strain 922 containing pCIB137 were also shown to producepyrrolnitrin, chitinase, and cyanide, but they did not display a changein morphology. Thus, it was shown that the introduction of pCIB 137 intoPseudomonas fluorescens strains (e.g. strain 914 and strain 922) whichare ineffective in the in vitro or in vivo inhibition of Rhizoctoniasolani, unexpectedly activated expression of previously inactive,undetected chitinase, cyanide, and pyrrolnitrin genes, and convertedthese strains into effective biocontrol agents in greenhouse assays.Also, in the case of strain 914, introduction of fragment E11unexpectedly caused a conversion in colony morphology to one verysimilar to that listed for P. fluorescens strain 915 in Table 1.

Example 8

Intact ORF 5 is necessary for gene activation

DNA sequence analysis revealed the presence of two NaeI recognitionsites, located 72 bp apart, within ORF5. Removal of the intervening DNAreduces the predicted gene product by 24 amino acids and may render thegene product nonfunctional. The 2 kb XhoI fragment was cloned into theXhoI site of the plasmid p SP72 (Promega). The resulting construct,designated pCIB138, contains no other NaeI sites besides the two inORF5. Digestion of pCIB138 DNA with NaeI followed by ligation at aconcentration of 30 ng/μl and transformation into E. coli strain S17-1yielded pCIB150, which contains the desired 72 bp deletion. The presenceof the deletion was confirmed by DNA sequencing. The ORF5-containingXhol fragment from pCIB150 was cloned into PVK100 to yield pCIB149.pCIB149 is identical to pCIB137 except for the deletion of the 72 bp ofDNA. When pCIB149 was introduced into strains 914 and 922 by conjugationas described in example 5, pyrrolnitrin, chitinase, or cyanideproduction was not detected. Thus, an intact ORF5 is necessary for geneactivation in bacterial strains.

Example 9

Gene-replacement experiment

The effect of ORF5 on gene regulation in strain 915 was demonstrated bythe following gene-replacement experiment. The fight 6.8 kb of fragmentE11 (bounded by fight-most EcoRI Is and BamHI sites, see FIG. 3) wascloned into pBR322 (digested with EcoRI and BamHI) to form pBREB6.8. The2kb XhoI fragment containing ORF 5 was removed from pBREB6.8 bydigestion with XhoI, then self-ligation to form pCIB139. A kanamycinresistance marker was introduced into pCIB139 by substituting theHindIII-SalI kanamycin resistance fragment from Tn5 for the tetracyclineresistance region bounded by HindIII and SalI in pCIB139. The resultingplasmid, pCIB 154, was used to receive the 2 kb XhoI fragment (with the72 bp NaeI deletion) from pCIB150 to form pCIB156. This plasmid wastransformed into the Escherichia coli strain S17-1 (Simon et al. (1983)BioTechnology 1:784-791 ), then introduced into strain 915 byconjugation, selecting for kanamycin resistance. Since pCIB156 cannot bemaintained autonomously in strain 915, the kanamycin resistanttransconjugants contained the plasmid, and its kanamycin resistantdeterminant, integrated into the chromosome. The most frequentintegration events took place by homologous recombination and at theregion of homology provided by the 2 kb XhoI fragment and itssurrounding sequences. Such transconjugants contain two copies of the 2kb XhoI region, one wildtype and one with the 72 bp NaeI deletion. Suchduplications are unstable in bacteria with a proficient homologousrecombination system and are lost at detectable frequenciesspontaneously when selective conditions favoring their formation areremoved. One such transonjugant is cultured in liquid medium withoutkanamycin selection and then plated on solid agar medium to obtainindividual colonies, again without kanamycin selection. Individualcolonies were tested for kanamycin sensitivity. Such colonies wereobtained at approximately 2% of the total. These kanamycin sensitivecolonies fall into two morphological classes, one resembling thewildtype, the other resembling the pleiotropic mutants. Southernhybridization results confirmed that both classes of colonies had lostthe integrated plasmid and that the class with the wildtype morphologycontained an intact 2 kb XhoI region, while the other class contains asmaller XhoI region corresponding to that with the 72 bp NaeI deletion.Colonies of the former class were identical to strain 915. Colonies ofthe latter class were identical to strain 915 except for a 72 bp NaeIdeletion in ORF 5. These derivatives of strain 915 no longer producepyrrolnitrin, chitinase, or cyanide. Thus, an intact ORF 5 is necessaryfor gene activation in strain 915.

Example 10

The native promoter of ORF 5 is contained within the 2 kb XhoI fragment

The following evidence indicates a promoter element directingtranscription of ORF 5 is likely to be found within the 2 kilobase XhoIfragment. A version of the broad host range plasmid pVK100 containingthe 2 kb XhoI fragment was isolated with the 2 kb insert in the oppositeorientation from that found in pCIB137. This plasmid was designatedpCIB151. Introduction of pCIB151 into pleiotropic mutants and into P.fluorescens strain 914 activated gene expression. The ability of the 2kb XhoI fragment to activate gene expression in each orientation, andthe previously described requirement of functional ORF 5 gene productfor gene activation, made it likely that transcription of ORF 5 relieson the presence of a promoter located within the 2 kb insert.Furthermore, Moolenar et al., (1987) have identified a promoterdirecting transcription of pairs upstream of the uvr-23 structural gene.At a nearly identical position upstream of ORF 5, we have identified thesequence TTGTCA-17bp-TTTTTT which is similar to the sigma-70 promoterconsensus sequence described by Rosenberg and Court, Annu. Rev. Genet.13:319-353 (1979). The sequence of a 983 base pair region containing theORF 5 structural gene, the possible promoter region, and the 5' end ofORF 4 (which is homologous to uvrC of E. coil) is presented in SEQUENCEID 3. The coordinates of these elements in sequence ID 3 are as follows:

Sequence with promoter homology: 23-51

ORF 5 structural gene: 99-740

5' end of ORF 4 (uvrC) 743-983

It is likely that a promoter native to the 2 kb XhoI fragment wasdirecting transcription of ORF 5 when ORF 5 was introduced intopleiotropic mutant derivatives of P. fluorescens 915 or into P.fluorescens strains 914 and 922. However, to express the ORF 5 class oftranscriptional activators in some bacterial strains, one skilled in theart will recognize that it might be beneficial to operably link thestructural ORF 5 class gene with a promoter and/or ribosome binding sitewhich functions more efficiently in the desired host bacterial genus toactivate latent genes. For example, to activate latent genes in Bacillusspecies with ORF 5 class genes, the ORF 5 class gene may be operablylinked to a Bacillus regulatory region. Such regulatory regions arereadily available to those skilled in the art. One possible method foraccomplishing this fusion between bacterial regulatory sequences and ORF5-class structural genes involves use of the overlap extensionpolymerase chain reaction strategy (Horton et al., Gene 77:61).

Example 11

Analysis of the ORF 5 gene.

a) The ORF 5 coding region is capable of encoding a 213 amino acidprotein with features of a bacterial transcription activator. Forexample, there is strong homology between domains 1 and 2 oftranscriptional regulators reviewed by Albright et al. (1989) andcomparable regions in ORF 5. The predicted aspartic acid residue atposition 54 of the protein lines up with the conserved aspartic acidresidues of other transcriptional activators of this class. It is theaspartic acid at this position which is typically phosphorylated byinteraction with a sensor component protein. Alignment of ORF 5 withuvr-23 of E. coli and with gacA of P. fluorescens CHA0 leads to theconclusion that ORF 5 contains the unusual translation start codon TTG,which is less efficient than either ATG or GTG start codons. It is worthnoting that at amino acid position 49 of ORF 5 resides an aspartic acidresidue, while a tyrosine residue is present at the equivalent positionof gacA. In virG, an Agrobacterium tumefaciens transcriptionalactivator, an asparagine to aspartic acid substitution near theconserved phosphorylation site converted virG to a constitutivetranscription activator which presumably no longer requiredphosphorylation by a sensor component. It is possible that our ORF 5 issuch a constitutive activator by virtue of the substitution of asparticacid for tyrosine.

b) As noted above, a promoter directing transcription of ORF 5 likelyresides within the 2 kb XhoI fragment. It is possible to map thelocation of this promoter by, for example, a combination of S1 nucleasemapping (Aiba et al., J. Biol. Chem. 256:11905-11910 (1981)) and primerextension mapping (Debarbouille and Raibaud, J. Bacteriol. 153:1221-1227(1983)) as was done for a different Pseudornonas promoter by, forexample, Gaffney et al., J. Bacteriol. 172:5593-5601 (1990). Oncelocated, a DNA fragment containing this promoter can be operably linkedif desired to ORF 5-class activators either by ligation of theappropriate DNA restriction fragments or by the overlap extension primerextension method of Horton et al.

c) Bacterial regulatory elements can be obtained from various sourcesincluding commercially available vectors, bacterial regulatory elementsknown in the art, and bacterial regulatory elements identified usingpromoterless marker-containing transposons, or promoter selectionvectors such as pKK175-6 and pKK232-8 (Pharmacia, Piscatoway, N.J.).Commercially available bacterial regulatory elements are available froma number of sources such as the plasmid expression vectors pKK233-2,pDR540, pDR720, pYEJ001, pPL-lambda (Pharmacia), or pGEMEX expressionvectors (Promega Biotec, Madison, Wis.). Bacterial regulatory elementsknown in the art include any bacterial regulatory element that is knownto function as a promoter, enhancer, ribosome binding site, and/or anyother regulatory control mechanism of the associated coding DNAsequence. An associated coding DNA sequence is a DNA sequence that isadjacent or adjoining 3' to the regulatory elements and which codes fora protein when transcribed and translated. Appropriate bacterialelements include those of Deretic et al., Bio/Technology 7:1249-1254(1989); Deuschle et al., EMBO J. 5:2987-2994 (1986); Hawley and McClure,Nucleic Acids Res. 11:2237-2255 (1983); Rosenberg and Court, Annu. Rev.Genet. 13:319-353 (1979), and references cited therein. Likewise,promoters for use in gram positive microorganisms such as Bacillusspecies are readily accessible to those skilled in the art. Any of theabove can be synthesized using standard DNA synthesis techniques.Bacterial regulatory elements include hybrid regulatory regionscomprising mixtures of parts of regulatory elements from differentsources. For example, the trp/lac (trc) promoter of pKK232-2 (Pharmacia)which combines the -35 region of the E. coli tryptophan operon promoterwith the -10 region of the E. coli lac operon promoter functionseffectively in Pseudomonas (Bagdasarian et al., Gene 26:273-282 (1983).

Certain bacterial promoters have the capability of functioningefficiently in a variety of bacterial genera. For example, promoters forselectable markers on the broad-host-range plasmid RSF1010 are known tofunction in at least the following bacterial genera: Acetobacter,Actinobacillus, Aerobacter, Aeromonas, Agrobacterium, Alcaligenes,Azotobacter, Azospirillum, Caulobacter, Desulfovibrio, Erwinia,Escherichia, Gluconobacter, Hyphomicrobium, Klebsiella, Methylophilus,Moraxella, Paracoccus, Proteus, Pseudomonas, Rhizobium, Rhodobacter,Serratia, Xanthomonas, Vibrio, Yersinia, and Zymoraonas (Morales et al.,1990. In: Pseudomonas: Biotransformations, Pathogenesis, and EvolvingBiotechnology, (Silver, Chakrabarty, Iglewski, and Kaplan, eds.) pp.229-241.)

Example 12

Biocontrol efficacies of the pCIB 137-containing transconjugants ofstrains 914 and 922 were compared to their natural parents. Bacterialcultures were grown overnight in Luria broth at 28 C. Cells werepelleted by centrifugation, then resuspended in sterile water to anoptical density of 2.5 at 600 nm (approximately 2×10⁹ colony formingunits per ml.). Rhizoctonia solani was cultured on autoclaved millet,then dried and ground into powder. Soil was prepared by mixing equalparts of potting soil (Metro-mix 360), sand, and vermiculite. This wasused to fill 15 cm diameter pots. A 2 cm deep circular furrow with atotal length of 30 cm was formed at the perimeter of each pot. Tencotton seeds (stoneville 506) were placed in each furrow. R.solani-infested millet powder was sprinkled evenly over the seeds in thefurrows at the rate of 100 mg/pot, followed by the application of 20 mlof bacterial suspension for each pot. Water was added in place ofbacterial suspension in the unbacterized control. Each treatmentconsisted of four replicate pots for a total of 40 seeds per treatment.The plants were grown in an environmentally controlled chamber with aday/night temperature regime of 26/21 C. The plants were rated fordisease severity after 10 days. The results (Table 4) clearly indicatethat strains 914 and 922 provide no disease control, whereas theirpCIB137-containing transconjugants provided good control of R. solani incotton.

                  TABLE 4                                                         ______________________________________                                        (Stand 10 DAPa)                                                               Treatment                                                                              Rep1    Rep2   Rep3  Rep4 Mean  % Biocontrol                         ______________________________________                                        NP.NTb   9       10     9     9    9.25  100.0                                P.NTc    0       2      1     1    1.00  0.0                                  914      3       0      3     1    1.75  9.1                                  914(pCIB 149)                                                                          0       1      0     2    0.75  -3.0                                 914(pCIB 137)                                                                          5       4      5     7    5.25  51.5                                 922      1       1      1     3    1.50  6.1                                  922(pCIB 137)                                                                          8       10     7     6    7.75  81.8                                 ______________________________________                                         1 = Uninfested control designated 100% biocontrol; Infested control           designated 0% biocontrol                                                      a = Days After Planting                                                       b = No Pathogen, No Treatment (Uninfested control)                            c = Pathogen, No Treatment (Infested control)                            

Example 13

Multiple copies of a gafA gene isolated from the Pseudomonas fluorescensstrain 914 activate expression of latent genes in strain 914

The 2.0 kb XhoI fragment containing the gafA gene of P. fluorescensstrain 915 was labeled and hybridized with XhoI-digested total genomicDNA from P. fluorescens strain 914. A 2.0 kb XhoI fragment from strain914 which hybridized to the probe is cloned in pBluescript SK+ and DNAsequencing is performed to verify that the done contains a gafAhomologue. The DNA sequence of the gafA homologue in strain 914 ispresented in Table 5. The strain 914 gafA homologue differs from thestrain 915 gafA at nine nucleotide positions, but only one of thesenucleotide differences is predicted to generate an amino add changedifference in the two proteins (amino acid residue 182 is threonine inthe strain 915 Gala protein and isoleucine in the 914 GafA protein). Thestrain 914 gafA gene was subcloned into the broad-host-range plasmidpVK100 and the resulting recombinant plasmid was introduced byconjugation into strain 914. Whereas expression of the singlechromosomal gafA gene in strain 914 was not capable of activatingexpression of genes required for the synthesis of pyrrolnitrin,chitinase, and cyanide, strain 914 derivatives containing multipleplasmid copies of the 914 gafA gene did synthesize pyrrolnitrin,chitinase, and cyanide.

                                      TABLE 5                                     __________________________________________________________________________    gafA open reading frame from pCIB 3341                                        __________________________________________________________________________    1 TTGATTAGGG TGCTAGTGGT CGATGACCAT GATCTCGTTC GTACAGGTAT                      51 TACCCGAATG CTGGCTGACA TCGATGGCCT GCAAGTGGTC GGTCAGGCCG                     101 AGTCAGGGGA GGAGTCCCTG CTCAAGGCCC GGGAGTTGAA ACCCGATGTG                    151 GTCCTCATGG ACGTCAAGAT GCCCGGGATC GGCGGTCTTG AAGCCACGCG 201                CAAATTGTTG CGCAGTCACC CGGATATCAA AGTCGTGGCC GTCACCGTGT 251                    GTGAAGAAGA CCCGTTCCCG ACCCGCTTGC TGCAAGCCGG TGCGGCGGGT 301                    TACCTGACCA AAGGTGCGGG CCTCAATGAA ATGGTGCAGG CCATTCGCCT 351                    GGTGTTTGCC GGCCAGCGTT ACATCAGCCC GCAAATTGCC CAGCAGTTGG 401                    TGTTCAAGTC ATTCCAGCCT TCCAGTGATT CACCGTTCGA TGCTTTGTCC 451                    GAGCGGGAAA TCCAGATCGC GCTGATGATT GTCGGCTGCC AGAAAGTGCA 501                    GATCATCTCC GACAAGCTGT GCCTGTCTCC GAAAACCGTT AATATCTACC 551                    GTTACCGCAT CTTCGAAAAG CTCTCGATCA GCAGCGATGT TGAACTGACA 601                    TTGCTGGCGG TTCGCCACGG CATGGTCGAT GCCAGTGCCT GA(SEQ ID NO.                     __________________________________________________________________________    5)                                                                        

Example 14

Activation of latent gene expression with the E. coli uvr-23 gene

The E. coli uvr-23 gene (also designated uvrY) is likely a member of aclass of bacterial transcriptional activators, although no knownfunction has yet been assigned to it in E. coli. A DNA fragmentcontaining the uvr-23 gene was obtained by amplifying by the polymerasechain reaction (Mullis and Faloona, Methods in Enzymology 155:335-350(1987)) a ca. 1.1 kb portion of the widely available E. coli K12 strainAB1157 genome. Primers for the polymerase chain reaction were preparedbased upon the published sequence of uvr-23 (Sharma et al., NucleicAcids Res. 14:2301-2318 (1986)). Two PCR primer oligonucleotides,5'-GGCGGAGTATACCATAAG-3' and 5'-ATAAGCTTACCACCAGCATCGTAC-3' were (SEQ IDNO's 6 and 7, respectively) used in the amplification of the ca. 1.1 kbDNA fragment at a concentration of 1 uM each in a 50 ul reaction mixcontaining, in reaction buffer supplied by the PCR kit manufacturer(Perkin Elmer Cetus), the four deoxyribonucleotides (dATP, dCTP, dGTP,and dTTP; 200 uM with respect to each), approximately 100 ng of E. coliK12 strain AB1157 genomic DNA, and 1 unit of Taq DNA polymerase. Typicalamplification cycle times and temperatures were 94 C. for 1 min,followed by 45 C. for 1 min, followed by 72 C. for 1 min (30 cyclestotal). Amplified DNA fragments were digested with the restrictionendonuclease HindIII, ligated with HindIII-digested pLAFR3, abroad-host-range plasmid capable of replication in Pseudomonas(Staskawicz et al., J. Bacteriol. 169:5789-5794 (1987)), and used totransform E. coli. When a pLAFR3 derivative containing the E. coliuvr-23 gene was mobilized by conjugation into Pseudomonas fluorescensstrain 914, the uvr-23 gene activated expression of genes involved inthe production of cyanide, chitinase, and pyrrolnitrin. Optimalactivation in P. fluorescens strain 914 apparently depends uponexpression of uvr-23 from the lac promoter of pLAFR3.

Example 15

Further delimitation of the 2.0 kb XhoI fragment to identify thesmallest subunit that is functional

The fact that an E. coli ORF 5-like transcription activator couldactivate latent genes in P. fluorescens strain 914 (Example 12), coupledwith the fact that disruption of ORF 5 in the 2.0 kb XhoI fragmentabolishes the gene-activating ability of this fragment, indicates thatit should be possible to define a smaller DNA fragment than the 2 kbfragment with gene-activating ability, provided ORF 5 is intact andexpressed. Expression of ORF 5 can be directed either from its nativepromoter, from a vector promoter, or from a heterologous promoteroperatively joined to the ORF 5 coding region. Smaller DNA fragments areprepared from a template consisting of the cloned 2 kb XhoI fragmentessentially by the procedure described for isolating the uvr-23 genefrom E. coli in Example 5. Pairs of oligonucleotide primers are preparedfor use in polymerase chain reaction amplification reactions. A commonprimer annealing to the template downstream of ORF 5 is present in eachprimer pair, while the remaining primer of each pair anneals to asequence at a different distance upstream of ORF 5. DNA fragments fromamplification reactions are cloned in a broad-host-range plasmid such aspLAFR3for introduction into P. fluorescens. P. fluorescenstransconjugants are tested for activation of latent gene activities byassaying production of chitinase, cyanide, and pyrrolnitrin. Thesmallest fragment activating latent genes is identified.

Example 16

Formulations of antifungal compositions employing liquid compositions oftransformed P. fluorescens bacteria which produce antibiotic substanceinhibitory to the growth of R. solani as the active ingredient.

In the following examples, percentages of composition are given byweight:

    ______________________________________                                        1. Emulsifiable concentrates:                                                                    a         b      c                                         ______________________________________                                        Active ingredient  20%       40%    50%                                       Calcium dodecylbenzenesulfonate                                                                  5%        8%     6%                                        Castor oil polyethlene glycol ether                                                              5%        --                                               (36 moles of ethylene oxide)                                                  Tributylphenol polyethylene glycol                                                               --        12%    4%                                        ether (30 moles of ethylene oxide)                                            Cyclohexanone      --        15%    20%                                       Xylene mixture     70%       25%    20%                                       ______________________________________                                    

Emulsions of any required concentration can be produced from suchconcentrates by dilution with water.

    ______________________________________                                        2. Solutions:    a       b       c     d                                      ______________________________________                                        Active ingredient                                                                              80%     10%     5%    95%                                    Ethylene glycol monomethyl ether                                                               20%     --      --    --                                     Polyethylene glycol 400                                                                        --      70%     --    --                                     N-methyl-2-pyrrolidone                                                                         --      20%     --    --                                     Epoxidised coconut oil                                                                         --      --      1%    5%                                     Petroleum distillate                                                                           --      --      94%   --                                     (boiling range 160-190°)                                               ______________________________________                                    

These solutions are suitable for application in the form of microdrops.

    ______________________________________                                        3. Granulates:       a      b                                                 ______________________________________                                        Active ingredient    5%     10%                                               Kaolin               94%    --                                                Highly dispersed silicic acid                                                                      1%     --                                                Attapulgite          --     90%                                               ______________________________________                                    

The active ingredient is dissolved in methylene chloride, the solutionis sprayed onto the carrier, and the solvent is subsequently evaporatedoff in vacuo.

    ______________________________________                                        4. Dusts:            a      b                                                 ______________________________________                                        Active ingredient    2%     5%                                                Highly dispersed silicic acid                                                                      1%     5%                                                Talcum               97%    --                                                Kaolin               --     90%                                               ______________________________________                                    

Ready-to-use dusts are obtained by intimately mixing the carriers withthe active ingredient.

Example 17

Formulation of antifungal compositions employing solid compositions oftransformed P. fluorescens bacteria which produce antibiotic substanceinhibitory to the growth of R. solani as the active ingredient

In the following examples, percentages of compositions are by weight.

    ______________________________________                                        1. Wettable powders: a        b      c                                        ______________________________________                                        Active ingredient    20%      60%    75%                                      Sodium lignosulfonate                                                                              5%       5%     --                                       Sodium lauryl sulfate                                                                              3%       --      5%                                      Sodium diisobutylnaphthalene sulfonate                                                             --       6%     10%                                      Octylphenol polyethylene glycol ether                                                              --       2%     --                                       (7-8 moles of ethylene oxide)                                                 Highly dispersed silicic acid                                                                      5%       27%    10%                                      Kaolin               67%      --     --                                       ______________________________________                                    

The active ingredient is thoroughly mixed with the adjuvants and themixture is thoroughly ground in a suitable mill, affording wettablepowders which can be diluted with water to give suspensions of thedesired concentrations.

    ______________________________________                                        2. Emulsifiable concentrate:                                                  ______________________________________                                        Active ingredient     10%                                                     Octylphenol polyethylene glycol ether                                                                3%                                                     (4-5 moles of ethylene oxide)                                                 Calcium dodecylbenzenesulfonate                                                                      3%                                                     Castor oil polyglycol ether                                                                          4%                                                     (36 moles of ethylene oxide)                                                  Cyclohexanone         30%                                                     Xylene mixture        50%                                                     ______________________________________                                    

Emulsions of any required concentration can be obtained from thisconcentrate by dilution with water.

    ______________________________________                                        3. Dusts:          a      b                                                   ______________________________________                                        Active ingredient   5%     8%                                                 Talcum             95%    --                                                  Kaolin             --     92%                                                 ______________________________________                                    

Ready-to-use dusts are obtained by mixing the active ingredient with thecarriers, and grinding the mixture in a suitable mill.

    ______________________________________                                        4. Extruder granulate:                                                        ______________________________________                                        Active ingredient  10%                                                        Sodium lignosulfonate                                                                             2%                                                        Carboxymethylcellulose                                                                            1%                                                        Kaolin             87%                                                        ______________________________________                                    

The active ingredient is mixed and ground with the adjuvants, and themixture is subsequently moistened with water. The mixture is extrudedand then dried in a stream of air.

    ______________________________________                                        5. Coated granulate:                                                          ______________________________________                                        Active ingredient 3%                                                          Polyethylene glycol 200                                                                         3%                                                          Kaolin            94%                                                         ______________________________________                                    

The finely ground active ingredient is uniformly applied, in a mixer, tothe kaolin moistened with polyethylene glycol. Non-dusty coatedgranulates are obtained in this manner.

    ______________________________________                                        6. Suspension concentrate:                                                    ______________________________________                                        Active ingredient         40%                                                 Ethylene glycol           10%                                                 Nonylphenol polyethylene glycol                                                                         6%                                                  (15 moles of ethylene oxide)                                                  Sodium lignosulfonate     10%                                                 Carboxymethylcellulose    1%                                                  37% aqueous formaldehyde solution                                                                       0.2%                                                Silicone oil in 75% aqueous emulsion                                                                    0.8%                                                Water                     32%                                                 ______________________________________                                    

The finely ground active ingredient is intimately mixed with theadjuvants, giving a suspension concentrate from which suspensions of anydesire concentration can be obtained by dilution with water.

Example 18

Isolation of further pleiotropic mutants

The transposon TnCIB116 is introduced into strain 915 by conjugation(Lam et al. (1990) Plant Soil 129:11-18). A collection of transposoninsertion mutants of strain 915 is obtained and screened for the loss ofpyrrolnitrin and chitinase production as described in Example 4. Afterscreening 10,000 transposon mutants, seven pleiotropic mutants which nolonger produce pyrrolnitrin, chitinase, or cyanide were obtained.

Example 19

Two genetic regions are required for gene activation in strain 915

The seven pleiotropic mutants fail into two genetic classes. pCIB137restored two of the seven transposon-induced mutants as well as mutant2-1, to wildtype phenotype, suggesting that the genetic defects in thesemutants were in ORF 5. Five mutants are not restored, indicating that atleast one other genetic locus is required for gene activation in strain915. A total gene library of strain 915 was introduced into thesemutants by conjugation and transconjugants which has regained wildtypemorphology were obtained. These transconjugants also producepyrrolnitrin, chitinase, and cyanide. The restoring clones were isolatedfrom these transconjugants. Restriction analysis indicates that theclones form an overlapping family of genetic fragments. The clonestested restored all five mutants of the second class to wildtypephenotype and had no effect on the two mutants of the first class. Theseclones define a second genetic region required for gene activation instrain 915. The smallest clone in the family, pCIB146, is analyzedfurther.

Example 20

Functional analysis of the second genetic region

The clone in pCIB146 was flanked by EcoRI sites (FIG. 4). There is oneinternal EcoRI site in the clone. The two EcoRI subclones were obtainedand tested for restoring ability. Neither one was able to restore mutantCGP 21, one of the five class II mutants, to wildtype phenotype,indicating that the internal EcoRI site defines a site critical to thefunctioning of the second genetic locus. There are two internal BamHIfragments in the clone. When these internal fragments were removed(pCIB191), the restoring ability was not affected. Finally, a 6 kbsubclone containing only the region from the internal HindIII site tothe leftmost internal BamHI site (pCIB168) retained restoring ability.Two clones were deposited which were able to complement the mutant: pCIB146 (about 25 kb) and pCIB 168 (about 6 kb). pCIB146 was deposited withNRRL on Jul. 29, 1993, and assigned accession number NRRL B-21118.pCIB168 was deposited with the NRRL on Jul. 29, 1993, and assignedaccession number NRRL B-21117.

Example 21

The second genetic region contains a gene homologous to lemA

DNA sequences surrounding the internal EcoRI site in pCIB146 wereobtained. Comparison against sequences contained in the GenBank databaserevealed significant homology with the lemA gene of Pseudomonas syringaepv. syringae strain B728a (Hrabak and 1992), a gene in the sensor familyof two-component regulatory systems. See Table 6.

                  TABLE 6                                                         ______________________________________                                        Comparison of pCIB168 with the published lemA sequence by Hrabak              et al. (1992) J. Bacterial. 174:3011-3020                                     lemA sequence                                                                 coding start GTG = 788                                                                          homology                                                    ______________________________________                                         788-1063         72%                                                         1101-1886         77%                                                         1616-1886         87%                                                         2182-2308         78%                                                         2528-2843         72%                                                         ______________________________________                                    

Example 22

Introduction of the lemA gene into Pseudomonas strains for therestoration of biocontrol functions

Clone pCIB146 is introduced into mutant Pseudomonas strains which lackbiocontrol functions due to an absence of the lemA gene. pCIB146 isintroduced into Pseudomonas strains from E. coli by conjugation and isfound to restore biocontrol functions, including chitinase, gelatinase,pyrrolnitrin and cyanide production.

Clone pCIB146 is also introduced into Pseudomonas strains which have noapparent defect in either the native lemA or gafA genes. An enhancementof biocontrol function, including production of chitinase, gelatinase,pyrrolnitrin and cyanide is found in the transformed strains by virtueof the increased lemA production in these strains overcoming alimitation in the capacity to phosphorylate the gafA protein.

The lemA gene is also introduced into Pseudomonas strains into which thegafA gene has already been introduced by the procedure described inExample 13. In this case the lemA gene is introduced on a plasmid whichutilizes an origin of replication different to pLAFR3 to enable bothgene constructions to be compatible in Pseudomonas. An enhancement ofbiocontrol function, including production of chitinase, gelatinase,pyrrolnitrin and cyanide is found in the strains expressing bothtransgenes by virtue of the increased lemA production in these strainsovercoming a limitation in the capacity to phosphorylate the gafAprotein, which in turn arises by virtue of the increased abundance ofgafA in the pseudomonad cells.

In any of the experimental approaches described above the lemA genecould be expressed behind a heterologous promoter, instead of from itsown promoter. Such a promoter would be required to be expressible inPseudomonas cells and may be expressed either constitutively or in aninducible fashion.

Example 23

Modification of the lemA gene to increase its kinase activity on gafA

By corollary with other sensor components it is assumed that lemAfunctions by interaction of the amino-terminal part of the protein withan unknown signal, autophosphorylation of a histidine located towardsthe carboxyterminus of the protein, which thus allows thephosphorylation of the gafA protein. The kinase activity on gafA isincreased using two experimental approaches.

First, the amino acid environment flanking the histidineautophosphorylation target is modified using PCR and cloning techniqueswell known in the art. Introduction of the modified lemA gene intoPseudomonas is achieved using a gene replacement technique (see example9), and the Pseudomonas strains thus modified are assessed againstnonmodified strains for chitinase, gelatinase, pyrrolnitrin, and cyanideproduction. Constructions which have a modified amino acid environmentadjacent to the target histidine which render the histidine a bettertarget for autophosphorylation also phosphorylate gafA more efficientlyand thus produce elevated levels of chitinase, gelatinase, pyrrolnitrinand cyanide.

Second, the amino-terminal sensor part of the lemA gene is modified bydeletion/substitution of amino acids using PCR and cloning techniqueswell known in the art. A series of modified constructions thus preparedare introduced into Pseudomonas strains using gene replacementtechniques (see example 9), and the Pseudomonas strains thus modifiedare assessed against non-modified strains for chitinase, gelatinase,pyrrolnitrin, and cyanide production. Constructions which have amodified sensor domain are able to autophosphorylate the targethistidine without the necessary interaction of the signal and maytherefore phosphorylate gafA more efficiently and thus produce elevatedlevels of chitinase, gelatinase, pyrrolnitrin and cyanide.

Example 24

Modification of the gafA gene to increase the efficiency ofphosphorylation of the protein

The amino acid environment flanking the presumed receiver domain of thegafA protein (around residue 54) is modified using PCR and cloningtechniques. A series of modified constructions thus prepared areintroduced into Pseudomonas strains using gene replacement techniques(see example 8b), and the Pseudomonas strains thus modified are assessedagainst non-modified strains for chitinase, gelatinase, pyrrolnitrin,and cyanide production. Constructions which have a modified receiverdomain and which are more readily phosphorylated produce elevated levelsof chitinase, gelatinase, pyrrolnitrin and cyanide.

Example 25

Pseudomonas strains carrying improved lemA and gacA genes

lemA and gafA modifications described in Examples 19 and 20 which whenintroduced into Pseudomonas cause a phenotype of elevated production ofchitinase, gelatinase, pyrrolnitrin and cyanide are combined into thesame Pseudomonas strain by remodifying the improved lemA-carrying strainby repeating the gene replacement experiment with the improved gafAconstruction.

Example 26

Modification of gafA to render the protein phosphorylation independent

The gafA gene is modified so as to render the gafA proteinphosphorylation independent. Since phosphorylation of the activatorcomponent of bacterial two-component regulatory systems leads to aconformational change in the DNA-binding domain of the activator, anyspecific amino acid substitutions, insertions, or deletions which leadto an equivalent conformational change render the activatorphosphorylationindependent. The use of a phosphorylation-independentversion of gafA in the activation of latent genes in bacterial strainsremoves the requirement that a strain contain an active version of LemAor an equivalent kinase.

The amino acid environment within the N-terminal half of gafA ismodified using PCR and cloning techniques well known in the art. Suchmutagenized versions of the gafA gene are cloned into broad-host-rangeplasmids and introduced into a lemA-mutant derivative of strain 915.Since introduction of the unaltered version of gafA into the lemA-mutantfails to restore synthesis of chitinase, pyrrolnitrin, cyanide, andgelatinase (see example 15), any altered versions of the GafA proteinwhich do restore some level of synthesis of these compounds in thelemA-strain are locked into a constitutively active conformation (i.e.phosphorylationindependent).

Example 27

Cloning Antipathogenic Biosynthetic Genes by Exploiting Regulators whichControl the Expression of the Biosynthetic Genes

Regulators such as gafA, lemA and rpoS may be used to clone biosyntheticgenes whose expression they control in the following manner. A libraryof transposon insertion mutants is created in a strain of microorganismwhich lacks the regulator or has had the regulator gene disabled byconventional gene disruption techniques. The insertion transposon usedcarries a promoter-less reporter gene (e.g. lacZ). Once the insertionlibrary has been made, a functional copy of the regulator gene istransferred to the library of cells (e.g. by conjugation orelectroporation) and the plated cells are selected for expression of thereporter gene. Cells are assayed before and after transfer of theregulator gene. Colonies which express the reporter gene only in thepresence of the regulator gene are insertions adjacent to the promoterof genes regulated by the regulator. Assuming the regulator is specificin its regulation for APS-biosynthetic genes, then the genes tagged bythis procedure will be APS-biosynthetic genes.

These genes can then be cloned and further characterized using standardtechniques well known in the art.

Example 28

Use of the gafA Regulator Gene for the Cloning of PyrrolnitrinBiosynthetic Genes from Pseudomonas

Pyrrolnitrin is an phenylpyrole compound produced by various strains ofPseudomonas fluorescens. P. fluorescens strains which producepyrrolnitrin are effective biocontrol strains against Rhizoctonia andPythium fungal pathogens (WO 94/01561). The biosynthesis of pyrrolnitrinis postulated to start from tryptophan (Chang et al., J. Antibiotics34:555-566 (1981)).

The gene cluster encoding pyrrolnitrin biosynthetic enzymes was isolatedusing the basic principle described in example 27 above. The regulatorgene used in this isolation procedure was the gafA gene from Pseudomonasfluorescens and is known to be part of a two-component regulatory systemcontrolling certain biocontrol genes in Pseudomonas. The gafA gene isdescribed in detail in pending application 08/087,636 which is herebyincorporated by reference in its entirety and in the publishedapplication WO 94/01561. gafA is further described in Gaffney et al.(Mol. Plant Microbe Int. 7:455-463 (1994)); also hereby incorporated inits entirety by reference) where it is referred to as "ORF5". The gafAgene has been shown to regulate pyrrolnitrin biosynthesis, chitinase,gelatinase and cyanide production. Strains which lack the gafA gene orwhich express the gene at low levels (and in consequence gafA-regulatedgenes also at low levels) are suitable for use in this isolationtechnique.

The transfer of the gafA gene from strain 915 to closely relatednon-pyrrolnitrin producing wild-type strains of Pseudomonas fluorescensresults in the ability of these strains to produce pyrrolnitrin.(Gaffney et al., Mol. Plant Microbe Int. 7:455-463 (1994)); see alsoHill et al. Applied And Environmental Microbiology 60 78-85 (1994)).This indicates that these closely related strains have the structuralgenes needed for pyrrolnitrin biosynthesis but are unable to produce thecompound without activation from the gafA gene. One such closely relatedstrain, strain 914, was used for the identification of the pyrrolnitrinbiosynthesis genes. The transposon TnCIB116 (Lain, New Directions inBiological Control: Alternatives for Suppressing Agricultural Pests andDiseases, pp 767-778, Alan R. Liss, Inc. (1990)) was used to mutagenizestrain 914. This transposon, a Tn5 derivative, encodes kanamycinresistance and contains a promoterless lacZ reporter gene near one end.The transposon was introduced into strain 914 by conjugation, using theplasmid vector pCIB116 (Lam, New Directions in Biological Control:Alternatives for Suppressing Agricultural Pests and Diseases, pp767-778, Alan R. Liss, Inc. (1990)) which can be mobilized into strain914, but cannot replicate in that organism. Most, if not all, of thekanamycin resistant transconjugants were therefore the result oftransposition of TnCIB116 into different sites in the strain 914 genome.

When the transposon integrates into the bacterial chromosome behind anactive promoter the lacZ reporter gene is activated. Such geneactivation can be monitored visually by using the substrate X-gal, whichreleases an insoluble blue product upon cleavage by the lacZ geneproduct. Kanamycin resistant transconjugants were collected and arrayedon master plates which were then replica plated onto lawns of E colistrain S17-1 (Simon et al., Bio/techonology 1:784-791 (1983))transformed with a plasmid carrying the wide host range RK2 origin ofreplication, a gene for tetracycline selection and the gafA gene. E colistrain S17-1 contains chromosomally integrated tra genes for conjugaltransfer of plasmids. Thus, replica plating of insertion transposonmutants onto a lawn of the S17-1/gafA E. coli results in the transfer tothe insertion transposon mutants of the gafA-carrying plasmid andenables the activity of the lacZ gene to be assayed in the presence ofthe gafA regulator (expression of the host gafA is insufficient to causelacZ expression, and introduction of gafA on a multicopy plasmid is moreeffective). Insertion mutants which had a "blue" phenotype (i.e. lacZactivity) only in the presence of gafA were identified. In thesemutants, the transposon had integrated within genes whose expressionwere regulated by gafA.

The ability to produce cyanide, chitinase, and pyrrolnitrin areactivities known to be regulated by gafA (Gaffney et al., Mol. PlantMicrobe Int. 7:455-463 (1994). The mutants described above (withintroduced gafA) were assayed for their ability to produce cyanide,chitinase, and pyrrolnitrin as described in Gaffney et al., Mol. PlantMicrobe Int. 7:455-463 (1994)). One mutant did not produce pyrrolnitrinbut did produce cyanide and chitinase, indicating that the transposonhad inserted in a genetic region involved only in pyrrolnitrinbiosynthesis. DNA sequences flanking one end of the transposon werecloned by digesting chromosomal DNA isolated from the selected insertionmutant with XhoI, ligating the fragments derived from this digestioninto the XhoI site of pSP72 (Promega, cat. #P2191 ) and selecting the E.coli transformed with the products of this ligation on kanamycin. Theunique XhoI site within the transposon cleaves beyond the gene forkanamycin resistance and enabled the flanking region derived from theparent STRAIN 914 strain to be concurrently isolated on the same XhoIfragment. In fact the Xhol site of the flanking sequence was found to belocated approximately 1 kb away from the end on the transposon.

A subfragment of the cloned XhoI fragment derived exclusively from the˜1 kb flanking sequence was then used to isolate the native (i.e.non-disrupted) gene region from a cosmid library of strain 915. Thecosmid library was made from partially Sau3A digested strain 915 DNA,size selected for fragments of between 30 and 40 kb and cloned into theunique BamHI site of the cosmid vector pCIB119 which is a derivative ofc2XB (Bates & Swift, Gene 26:137-146 (1983)) and pRK290 (Ditta et al.Proc. Natl. Acad. Sci. USA 77:7247-7351 (1980)). pCIB119 is a double-cossite cosmid vector which has the wide host range RK2 origin ofreplication and can therefore replicate in Pseudomonas as well as E.coli. Several clones were isolated from the strain 915 cosmid clonelibrary using the ˜1 kb flanking sequence as a hybridization probe. Ofthese one clone was found to restore pyrrolnitrin production to thetransposon insertion mutant which had lost its ability to producepyrrolnitrin. This clone had an insertion of ˜32 kb and was designatedpCIB 169.

Example 29

Isolation of Genes Encoding Resorcinol

Two transposon-insertion mutants have been isolated which lack theability to produce the antipathogenic substance2-hexyl-5-propyl-resorcinol which is a further substance known to beunder the global regulation of the gafA gene in Pseudomonas fluorescens(WO 94/01561 ). The insertion transposon TnCIB116 was used to generatelibraries of mutants in strain 915 and a gafA-derivative of strain 915(BL1826). The former was screened for changes in fungal inhibition invitro; the latter was screened for genes regulated by gafA afterintroduction of gafA on a plasmid (see Section C). Selected mutants werecharacterized by HPLC to assay for production of known compounds such aspyrrolnitrin and 2-hexyl-5-propyl-resorcinol. The HPLC assay enabled acomparison of the novel mutants to the wild-type parental strain. Ineach case, the HPLC peak corresponding to 2-hexyl-5-propyl-resorcinolwas missing in the mutant. The mutant derived from strain 915 isdesignated BL1846. The mutant derived from BL1826 is designated BL1911.

The resorcinol biosynthetic genes can be cloned from theabove-identified mutants in the following manner. Genomic DNA isprepared from the mutants, and clones containing the transposoninsertion and adjacent Pseudomonas sequence are obtained by selectingfor kanamycin resistant clones (kanamycin resistance is encoded by thetransposon). The cloned Pseudomonas sequence is then used as a probe toidentify the native sequences from a genomic library of P. fluorescensstrain 915. The cloned native genes are likely to represent resorcinolbiosynthetic genes.

Example 30

Identification and cloning of the rpoS gene encoding the alternate sigmafactor rpoS (sigma-38) from Pseudomonas fluorescens strain 915

In Escherichia coli, it has been demonstrated that an alternate sigmafactor designated rpoS is required for the transcription of a large setof genes which are preferentially expressed during stationary phaseconditions (i.e. conditions of nutrient stress) (Siegele and Kolter, J.Bacteriol. 174:345-348 (1992); Hengge-Aronis, Cell 72:165-168 (1993)).Antifungal factors involved in Pseudomonas biocontrol efficacy areactively expressed during stationary phase, creating the possibilitythat an rpoS homologue might exist in Pseudomonas. To investigate thispossibility, an internal portion of the E. coli rpoS gene was obtainedfor use as a hybridization probe by designing polymerase chain reactionprimers based on the published sequence of the E. coli rpoS gene (Mulveyand Loewen, Nucleic Acids Res. 17:9979-9991 (1989)) and altered to eachcontain a HindIII restriction site for subsequent cloning purposes. TwoPCR primer oligonucleotides, 5'-GGTCAAGCTTATGGGACAA-3' and5'-GAGAAGCTTGCGTCTGGTGG-3' were used at a concentration of 1 uM each ina 50 ul reaction mix containing in reaction buffer supplied by the PCRkit manufacturer (Perkin Elmer Cetus), the four deoxyribonucleotides(dATP, dCTP, dGTP, and dTTP; 200 uM with respect to each), approximately100 ng of E. coli K12 strain AB1157 genomic DNA, and 1 unit of Taq DNApolymerase. Typical amplification cycle times and temperatures were 94C. for 1 min, followed by 45 C. for 1 min, followed by 72 C. for 1 min(30 cycles total). These conditions resulted in the amplification of aca. 265 base pair DNA fragment which was digested with HindIII andcloned into the HindIII site of pBluescript SK II+ (Stratagene). DNAsequence analysis of the ca. 265 base pair fragment confirmed that thesequence was an internal portion of the E. coli rpoS gene. Therecombinant plasmid containing the ca. 265 base pair fragment waslabeled by incorporating biotinylated dUTP in a random priming reactionas described by the manufacturer of the Flash Prime-IT II labeling anddetection system (Stratagene, La Jolla, Calif.). The labeled plasmid wasused as a hybridization probe against a preparation of genomic DNAisolated from Pseudomonas fluorescens strain 915 which had been digestedwith EcoRI, electrophoresed through a 0.7% agarose gel, and transferredby capillary blotting to a nylon membrane. A single hybridization bandwas observed, corresponding to a genomic EcoRI fragment of approximately9 kb. Plasmid DNA preparations were prepared from pools consisting of 60colonies each obtained from a cosmid library of P. fluorescens strain915 DNA, digested with EcoRI, electrophoresed through a 0.7% agarosegel, and transferred to a nylon membrane. The rpoS gene probe hybridizedwith a ca. 7 kb EcoRI fragment present from one such pooled preparation.This fragment likely represented a portion of the 9 kb genomic fragmentwhich had previously hybridized, with one EcoRI site belonging to thecosmid vector. The 7 kb EcoRI fragment was subcloned from the cosmidvector into the EcoRI site of pBluescript SK II+. This plasmid wasdesignated pCIB3360 and was deposited on Aug. 8, 1994, an E coli hostwith the NRRL (Agricultural Research Service Culture Collection, Peoria,Ill.) and assigned NRRL accession number B-21299. DNA sequence analysisrevealed that the 7 kB EcoRI fragment did in fact have the cosmid vectorEcoRI site as one endpoint and that this fragment contained the rpoSgene of P. fluorescens strain 915.

Example 31

Sequence of the P. fluorescens swain 915 rpoS gene

The DNA sequence of the P. fluorescens strain 915 rpoS gene and thededuced amino acid sequence of the RpoS sigma factor are provided inSequence ID No. 8 and Sequence ID No. 9, respectively. The deduced aminoacid sequence is greater than 86% identical to that of the unpublishedsequence of a Pseudomonas aeruginosa RpoS (Tanaka and Takahashi, GenBankaccession #D26134, (1994)). It is also highly homologous to the E. coli,Salmonella typhimurium, and Shigella flexneri RpoS sigma factors (Mulveyand Loewen, Nucleic Acids Res. 17:9979-9991 (1989), GenBank accession#U05011, GenBank accession #U00119).

Example 32

Requirement of a functional copy of RpoS for the expression ofbiocontrol factors and secondary metabolites

A kanamycin resistance cartridge is cloned as a BamHI fragment frompUC4-KIXX (Pharmacia) into the rpoS gene of P. fluorescens strain 915.An EcoRI-BamHI fragment (derived from the 7 kb EcoRI fragment)containing the 5' half of rpoS is subcloned in pBluescript SK II+adjacent to a BamHI-HindIII PCR amplification product consisting ofsequences immediately downstream of rpoS. This subclone is digested withBamHI to allow insertion of the kanamycin resistance cartridge. Thistagged defective version of rpoS is excised from pBluescript as an EcoRIfragment and cloned into the EcoRI site of the cloning vector pBR322.This pBR322 derivative is mobilized by conjugation into P. fluorescensstrain 915. Since pBR322 cannot replicate in Pseudomonas, the majorityof kanamycin resistant colonies result from recombination between rpoSor its flanking sequences and the homologous sequences in the P.fluorescens strain 915 chromosome. Double crossover events, in which thenormal version of rpoS is replaced by the disrupted version, aredetected as resulting in kanamycin resistant colonies which lack thepBR322 tetracycline resistance marker. Such events are confirmed bySouthern hybridization. The constructed P. fluorescens rpoS mutants aretested for the production of antifungal factors and secondarymetabolites such as pyrrolnitrin, chitinase, cyanide, and gelatinase andare found to be deficient or reduced in the production of these factors.

While the present invention has been described with reference tospecific embodiments thereof; it will be appreciated that numerousvariations, modifications, and embodiments are possible, andaccordingly, all such variations, modifications and embodiments are tobe regarded as being within the spirit and scope of the presentinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 642 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas fluorescens                                         (B) STRAIN: CGA267356                                                         (C) INDIVIDUAL ISOLATE: ORF 5                                                 (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pCIB137                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..639                                                          (D) OTHER INFORMATION: /transl_except= (pos: 1 .. 3, aa: Met)                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGATTAGGGTGCTAGTAGTCGATGACCATGATCTCGTTCGTACAGGT48                            MetIleArgValLeuValValAspAspHisAspLeuValArgThrGly                              151015                                                                        ATTACACGAATGCTGGCTGACATCGATGGCCTGCAAGTGGTCGGCCAG96                            IleThrArgMetLeuAlaAspIleAspGlyLeuGlnValValGlyGln                              202530                                                                        GCCGAGTCAGGGGAGGAATCCCTGCTCAAGGCCCGGGAGTTGAAACCC144                           AlaGluSerGlyGluGluSerLeuLeuLysAlaArgGluLeuLysPro                              354045                                                                        GATGTGGTCCTCATGGACGTCAAGATGCCCGGGATCGGCGGTCTTGAA192                           AspValValLeuMetAspValLysMetProGlyIleGlyGlyLeuGlu                              505560                                                                        GCCACGCGCAAATTGTTGCGCAGTCACCCGGATATCAAAGTCGTGGCC240                           AlaThrArgLysLeuLeuArgSerHisProAspIleLysValValAla                              65707580                                                                      GTCACCGTGTGTGAAGAAGATCCGTTCCCGACCCGCTTGCTGCAAGCC288                           ValThrValCysGluGluAspProPheProThrArgLeuLeuGlnAla                              859095                                                                        GGCGCGGCGGGTTACCTGACCAAGGGGGCGGGCCTCAATGAAATGGTG336                           GlyAlaAlaGlyTyrLeuThrLysGlyAlaGlyLeuAsnGluMetVal                              100105110                                                                     CAGGCCATTCGCCTGGTGTTTGCCGGCCAGCGTTACATCAGCCCGCAA384                           GlnAlaIleArgLeuValPheAlaGlyGlnArgTyrIleSerProGln                              115120125                                                                     ATTGCCCAGCAGTTGGTGTTCAAGTCATTCCAGCCTTCCAGTGATTCA432                           IleAlaGlnGlnLeuValPheLysSerPheGlnProSerSerAspSer                              130135140                                                                     CCGTTCGATGCTTTGTCCGAGCGGGAAATCCAGATCGCGCTGATGATT480                           ProPheAspAlaLeuSerGluArgGluIleGlnIleAlaLeuMetIle                              145150155160                                                                  GTCGGCTGCCAGAAAGTGCAGATCATCTCCGACAAGCTGTGCCTGTCT528                           ValGlyCysGlnLysValGlnIleIleSerAspLysLeuCysLeuSer                              165170175                                                                     CCGAAAACCGTTAATACCTACCGTTACCGCATCTTCGAAAAGCTCTCG576                           ProLysThrValAsnThrTyrArgTyrArgIlePheGluLysLeuSer                              180185190                                                                     ATCAGCAGCGATGTTGAACTGACATTGCTGGCGGTTCGCCACGGCATG624                           IleSerSerAspValGluLeuThrLeuLeuAlaValArgHisGlyMet                              195200205                                                                     GTCGATGCCAGTGCCTGA642                                                         ValAspAlaSerAla                                                               210                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 213 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetIleArgValLeuValValAspAspHisAspLeuValArgThrGly                              151015                                                                        IleThrArgMetLeuAlaAspIleAspGlyLeuGlnValValGlyGln                              202530                                                                        AlaGluSerGlyGluGluSerLeuLeuLysAlaArgGluLeuLysPro                              354045                                                                        AspValValLeuMetAspValLysMetProGlyIleGlyGlyLeuGlu                              505560                                                                        AlaThrArgLysLeuLeuArgSerHisProAspIleLysValValAla                              65707580                                                                      ValThrValCysGluGluAspProPheProThrArgLeuLeuGlnAla                              859095                                                                        GlyAlaAlaGlyTyrLeuThrLysGlyAlaGlyLeuAsnGluMetVal                              100105110                                                                     GlnAlaIleArgLeuValPheAlaGlyGlnArgTyrIleSerProGln                              115120125                                                                     IleAlaGlnGlnLeuValPheLysSerPheGlnProSerSerAspSer                              130135140                                                                     ProPheAspAlaLeuSerGluArgGluIleGlnIleAlaLeuMetIle                              145150155160                                                                  ValGlyCysGlnLysValGlnIleIleSerAspLysLeuCysLeuSer                              165170175                                                                     ProLysThrValAsnThrTyrArgTyrArgIlePheGluLysLeuSer                              180185190                                                                     IleSerSerAspValGluLeuThrLeuLeuAlaValArgHisGlyMet                              195200205                                                                     ValAspAlaSerAla                                                               210                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5559 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas fluorescens                                         (B) STRAIN: CGA267356                                                         (C) INDIVIDUAL ISOLATE: 5.6 kb EcoRI- HindIII restriction                     fragment                                                                      (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pCIB137                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 210..1688                                                       (D) OTHER INFORMATION: /note= "ORF 1, transcribed left to                     right"                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1906..3633                                                      (D) OTHER INFORMATION: /note= "ORF 2, transcribed left to                     right"                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 4616..4691                                                      (D) OTHER INFORMATION: /note= "glyW, transcribed right to                     left"                                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 4731..5318                                                      (D) OTHER INFORMATION: /note= "ORF 3, transcribed right to                    left"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGATGACATGCCGCGCGCCGGCATCGACACGCAAATGGTCGACCTGGTGCTGCCG60                GTGGTCGAAATGCCGCAGAAGCTGCTGGAGCTGTGGCGCAACTCTCAGCTCATCACCCTG120               CCGACCGCCAACGATCCGCAAATCAAGGTCTCGGCGCCGGTGTCCAAACGCGATGCCGCG180               GCGGCGAACAGCAGCTGCAAGACATCCTGATGCTGTTGCGCACCGGCACCGGCCATGACT240               TCAAGCATTACAAGCGCGCCACGGTGCTGCGGCGGATCGAGCGCCCGCTGCAGGTCACCG300               CCCAGCCGGACCTCGCCGCCTATCACGATTACCTGCAGATGCACCCTGAAGAAACCAAGG360               CGCTGCTGGGCGACATGCTGATCGGCGTGACCAATTTCTTTCGCGACCGCGAGGCCTTCG420               AAGCCCTGGAGCGCAATGTCATTCCTGCCCTGGTGAAGTCCTTGCAGGACAGCCAACCGC480               ACCGTGAAGACGTGCGCATCTGGTCCGCCGGCTGCTCCACGGGTGAAGAGGCCTATAGCC540               TGGCAATCGTCGCCAGCGAGCAGATGGCCCTGGAGGCCTGCAACGCCAAGCTGCAGGTAT600               TCGCGACCGATATCGACGATCGTGCCATCGCCCAGGGACGCAAGGGGGTCTATCCCGAAG660               CGATCGTTACCGATGTGCCTCCGCAGCGCATGCGCCAGTACTTTTCCCGGGAAAACCAGC720               ATTACCGGGTGCGCAAGGAGATTCGCGAAAAGGTGCTGTTCGCCAAGCACAGCCTGCTGG780               CGGATCCGCCATTTTCGCAGATCGACTTGATCGTCTGCCGTAACCTGCTGATCTACCTGG840               ACCGCGACGTGCAACGGGAGATCCTGCAGATGTTCCACTTCGCCCTGCGTCCTGGAGGCT900               ACCTGTTCCTCGGTTCCTCCGAATCCGCGGACGGCTGCCAGGATCTGTTCGTGCCGGTCG960               ACAAGCGCAACCGCATTTTCCGGGTACGGCCCAACTCGGCCACGGTTCGCCGCGCGCCCA1020              CCATGCCGCGACGGCGTACATGCGCACCATCGGCAGCCCCCACCCCGTGGAAACCAAGTG1080              TCTCGCGCAAAACCTCGTTCGCCGACATCCACCTTCGCGCCCTGGAAAAGTGCGCGCCGC1140              CGAGCATGATCGTCGATGCCAACGCCGACATCCTGCACATGAGCGAAGGCGCCGGCCGGT1200              TCCTGCGCTATGTCGCGGGGGAAATCACCCGCAACCTGCTGACCCTGATCCAGCCCGAGC1260              TGCGCCTTGAACTGCGCACCACGCTGTTCCAGGTGCAACAGTCCGGTGTTGCGGTGACCG1320              CCGCCGGGTGCGCATCGAGCGGGAAAAGAAGCCTTGTTTCATCGACCTCACAGCCCGCCC1380              CTTCAAGGACGAGGAAACCGACAACGAATATGTGCTGGTGGTGTTCGAGGAGACCGAGGC1440              CGACCCACGGGAGCTGCGCGAGACCAGCGCCAGCCAGACGGAAAACCAGATGCTGGCCAA1500              CCTCGAGCGGGAGTTGCAGCGGACCAAATTGCACCTGCAGGACACCATCGAGCAATCGGA1560              AGTCTCCAGCGAGGAGCTCAAGGCGTCGAACGAAGAAATGCAGGCGCTCAATGAAGAGCT1620              GCGCTCGGCCACCGAAGAGCTGGAAACCAGCAAGGAAGAGTTGCAGTCGATCAATGAAGA1680              GCTGCTGACGGTCAATTACGAGCTGAAAACCAAGGTCGAGGAAACCGACAAGATCAACGA1740              CTACCTGACCAACCTGATCGCCTCCACCGACATCGCCACGGTGTTCGTCGACCGCAACAT1800              GCGCATCCGCTGGTTCACCCCGCGCGCCACCGACATTTTCAGCATGCTGCCGGTGGACAC1860              CGACGCTCATTACTGGACATCACCCACCGCCTGAACTACCCGGAAATGGCCGAGGACGCC1920              GCGACCGTGTTCGAGTCGTTGAGCATGATCGAGCGTGAAGTCAACAGCGACGATCAGCGC1980              TGGTACATCGCACGCCTGTTGCCCTATCGCTCCAGCGAAGACCATATCGACGGCACCGTG2040              CTGACCTTCATCGATATCACCAAGCGCCGGCTGGCCGAGGAGGAACTGCGCCTGGGCGAA2100              GAACGCATGCGCCTGGTCGCCGAAAGCACCCATGATTTCGCCATCATCATCCTCGACAAC2160              CAGGGCCTCATCACCGACTGGAACACCGGGGCGCAACTGATCTTCGGCTATACCAAGGAC2220              GAAGTGCTGGGCGCCTATTACGACCTGATTTTCGCGCCTGAGGACCGCGCCGGCGGCGTG2280              CCGGAAAGCGAGCTGCTCACCGCCCGCGAACACGGCCGCAGCGACGATGAACGCTGGCAT2340              ATACGCAAGGACGGCGAGCGCTTTTTCTGCAGCGGCGAAGTCACGCGGCTCAAGGGTGAC2400              AGCCTGCAAGGCTACGTGAAAATAGCCCGCGACCTGACGGGCCACAAACGCATGCAGGAC2460              GAGCAGAACCAGAAGCTGATGGAGACCCAGACCCACAGCCACCTCAAGGATGAGTTTTTC2520              GCGGTGATGTCCCATGAACTCAAGCATCCGCTCAACCTGATCCAGCTCAACGCCGAGTTG2580              CTGCGTCGCCTGCCGACGACCAAGGCGGCCGCCCCTGCCCTCAAGGCGGTCAATACCATT2640              TGCGAGGCTGTCTCCAGCCAGGCGCGGATCATCGACGACCTGCTGGATGTGCGGCGTTTG2700              CGCACCGGCAAGCTCAAGCTGAAGAAACAGCCGGTGGATCTTGGCCGGATCCTGCAGGAC2760              ATCCATACCGTGGTGCTCAGCGAAGGGCATCGCTGCCAGGTGACGCTGCAAGTGCCGTTG2820              CCACCGCAACCGCCGTTAATGATCGATGCCGATGCGACGCGGCTGGAGCAGGTGATCTGG2880              AACCTGGTGAACAACGCCCTGAAATTCACCCCGGCCAATGGCTTGGTCCAGTTGATCGCC2940              CAGCGGGTCGAGGATAAGGCGCACGTGGATGTCATCGACAGCGGCGTGGGCCTGGCCGAG3000              GAAGACCAGAACAAGGTGTTCGACCTTTTCGGCCAGGCGGCCAACCAGCACGGCACTCAT3060              CAACGCGACGGGCTGGGCATCGGCCTGTCACTGGTGCGCCAGCTGGTGGAAGCCCACGGC3120              GGCTCGGTCAGCGTGCAGTCGAAGGGGCTGGGCCAGGGATGCACCTTTACCGTGCTCTTG3180              CCCCTGAGCCACCCCAACGACAGCGCTCCCAAACAGCCCGCGTCGCGGGGTGTCGAACGC3240              CTTGCCGGCATCAAGGTGCTGCTGGTGGACGACTCGCGGGAAGTCATGGAAGTCCTGCAA3300              CTGCTGCTGGAGATGGAGGGCGCGCAAGTCGAGGCCTTCCACGACCCGCTGCAGGCCTTG3360              GGCAATGCCAGGAACAACAGTTACGACCTGATCATTTCAGACATCGGCATGCCGATTATG3420              AACGGCTACGAACTGATGCAGAACCTGCGCCAGATCGCTCACCTGCACCATACGCCAGCG3480              ATTGCGCTGACCGGTTACGGCGCCAGCAGCGACCAGAAGAAGTCCCAGCATGCGGGATTC3540              GATCGGCATGTGAGCAAACCCGTGGCTCAGGACCCGCTGATCGACCTGATCAGGGAGCTG3600              TGCAGCCAGGGCTTGCGCTCGGCTGAGCACTGATGGTCTAGACCCGGCGAACCCACCTCG3660              TCGGCCTTGAGCGCGGCGAGCGCCATTGCCTGCTGGGCAGCTATTCACGCTTGCGGATCG3720              TCGCGCCTGCGGGCCACCGCCTCTTTGATGGCTTGCTCATAGGCGGCGTTGGCCTGGTCC3780              TTGAGCTTGAGCCAATCGTCCCAATCGATCACGCCGTTGCGCAGCAACTCCTCGGCCGCG3840              CTTAACAGCGCCTGATGCCAGGCGTCCGGCGAGCCGGAACGGTAGTCACGGTCTTCCAGC3900              AGGCCTTGCCAGGCGTCCAGTTCCGGTGTCTTGCGTTCATTGACCATGGCAGCCACGGCC3960              TTTGTTCATTGCCGATAAATCGGCGAGTGGGTGGTGGGTTTCTCGGATATGCGCCCTGTC4020              CTGCTCGAGAACGGCCAGGCCGGGACATTGCTCAACGGTCAGCGACCGGATGGAGCTCGA4080              GCGGCATGCCATCGACCAGCGTCAAGGTCAGGTTCTCGATGGTGCCGGCGATCCGGTCCT4140              TGAATACCGGTTCGCCGTCCGGATCCAACTCATCGTAGAAAAAGCGCGTGCCTTCGAGCC4200              AGCCAATGGTCGTTTGCAGGTCCGGCCCCAGGTAATACTTGCCGTCAAGGAAAAACCCGG4260              TAAAGGGCTCCACCCGCTCGCGATTCTCAATGACATAACGTATTCCAGCGTGCATACCTG4320              TCGATTTATCGAGCATGGCGTCGATCTCCCAGCAGATGAATCCGGTAGACCGCGTGGCTT4380              TTTCACTGTTCCTTTTGATTGCCCGCCCGACGCTGGCGAGCCTTGCTCGCGCGTCCTGGC4440              CGCATTGCGCGGCGAATGGGCGACGTCGAATCCGATCTGCAAGTGCCCAGCTAGCGGCCC4500              GGCCACGGCAATACGGGCTTCAGGTACGGCTTAGAAAGAAGAATGACGATTGGCTCGACA4560              TATTTTTTGGCGCAAAAAAAAATGGACCTCTTTTCAGAGGTCCATTTTTAATATTTGGAG4620              CGGGAAACGAGACTCGAACTCGCGACCCCGACCTTGGCAAGGTCGTGCTCTACCAACTGA4680              GCTATTCCCGCGTCTTGGTGGTGTGCATTTTATAGAAATTCGAAACTGCGTCAACCCCTT4740              GATTCAAAAAGTTTTATTTCTTTTCTACCATCGGTCTTCAGGTGCGGCCAGGCAGCGCGC4800              AGGTACTGCAACATCGACCACAGGGTCAGCCCTCCGGCGATCAGCAGGAAGGCATAACCC4860              AGCAGCACCCAGAAGGTGAAGGCCGGCGGATTGGCCAGCAGGATCACCAGCGCCAGCATC4920              TGCGCGGCAGTTTTCCGATTTGCCCATGTTGGACACCGGCCACCTGGGCGCGTGCGNCCG4980              AGCTCGGCCATCCACTCGCGAAGGGCGGACACCACGATTTCACGCCCGATGATCACCGCT5040              GCCGGCAGGGTCAGCCACAGGTTGCCGTGCTCTTGCACCAGCAGCACCAGGGCCACCGCC5100              ACCATCAACTTGTCGGCCACCGGATCGAGGAAGGCCCCGAACGGCGTGCTCTGCTCCAGA5160              CGCCGCGCCAGGTAGCCATCAAGCCAGTCGGTGGCCGCGGCGAACGCAAAGACGGAACTG5220              GCGGCCATGTAGCTCCAGTTGTAAGGCAGGTAAAACAGCAAAATGAAGATCGGGATGAGC5280              AGAACGCGTAGAACGGTGATCAGATTAGGGATATTCATCGGCACAACTGGCTACGAGGTG5340              AGTGGCAATCTACTCGGAAAAGACAGCAGATGAGGTAGCACGGCCATTCTACGGGCTTCT5400              GCCACAGCGTGTCTAACACTGTTCCAAGACTTCGGGCCGCTCGAAAGAGCAACTTCAGAA5460              GGTCTACACGCGCAAAATAAGACATTCAGTTCTTCTGTAAGTACCGTGTAGATCGGGATC5520              TATCAGCGGTGCCCCGCCAAAAAGGAAGCCTTGAAGCTT5559                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 983 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas fluorescens                                         (B) STRAIN: CGA267356                                                         (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pCIB137                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 23..51                                                          (D) OTHER INFORMATION: /note= "sequence with promoter                         homology"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 99..740                                                         (D) OTHER INFORMATION: /note= "ORF 5 structural gene"                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 743..983                                                        (D) OTHER INFORMATION: /note= "5'end of ORF 4 (uvrC)"                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCAAGTGCTTTTTATATGGTGTTTGTCATTAGGTCAGCACGCTGCTTTTTTGCTAAGGTG60                TCCGGCAACCTATAAGACCCAAATCGCGAGGTGTCTGCTTGATTAGGGTGCTAGTAGTCG120               ATGACCATGATCTCGTTCGTACAGGTATTACACGAATGCTGGCTGACATCGATGGCCTGC180               AAGTGGTCGGCCAGGCCGAGTCAGGGGAGGAATCCCTGCTCAAGGCCCGGGAGTTGAAAC240               CCGATGTGGTCCTCATGGACGTCAAGATGCCCGGGATCGGCGGTCTTGAAGCCACGCGCA300               AATTGTTGCGCAGTCACCCGGATATCAAAGTCGTGGCCGTCACCGTGTGTGAAGAAGATC360               CGTTCCCGACCCGCTTGCTGCAAGCCGGCGCGGCGGGTTACCTGACCAAGGGGGCGGGCC420               TCAATGAAATGGTGCAGGCCATTCGCCTGGTGTTTGCCGGCCAGCGTTACATCAGCCCGC480               AAATTGCCCAGCAGTTGGTGTTCAAGTCATTCCAGCCTTCCAGTGATTCACCGTTCGATG540               CTTTGTCCGAGCGGGAAATCCAGATCGCGCTGATGATTGTCGGCTGCCAGAAAGTGCAGA600               TCATCTCCGACAAGCTGTGCCTGTCTCCGAAAACCGTTAATACCTACCGTTACCGCATCT660               TCGAAAAGCTCTCGATCAGCAGCGATGTTGAACTGACATTGCTGGCGGTTCGCCACGGCA720               TGGTCGATGCCAGTGCCTGACAATGACCGACCCGTTTGATCCCAGTGCTTTTCTTTCCAC780               CTGCAGTGGCCGTCCTGGCGTGTATCGCATGTTCGACAGCGATACGCGTCTGCTGTACGT840               CGGTAAAGCCAAGAACCTGAAGAGCCGCCTGGCCAGCTACTTTCGCAAGACCGGCCTGGC900               GCCCAAGACCGCTGCCCTGGTGGGGCGCATCGCAGATCGAAACCACCATCACCGCCAACG960               AGACCGAAGCCCTGCTGCTCGAG983                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 642 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTGATTAGGGTGCTAGTGGTCGATGACCATGATCTCGTTCGTACAGGTATTACCCGAATG60                CTGGCTGACATCGATGGCCTGCAAGTGGTCGGTCAGGCCGAGTCAGGGGAGGAGTCCCTG120               CTCAAGGCCCGGGAGTTGAAACCCGATGTGGTCCTCATGGACGTCAAGATGCCCGGGATC180               GGCGGTCTTGAAGCCACGCGCAAATTGTTGCGCAGTCACCCGGATATCAAAGTCGTGGCC240               GTCACCGTGTGTGAAGAAGACCCGTTCCCGACCCGCTTGCTGCAAGCCGGTGCGGCGGGT300               TACCTGACCAAAGGTGCGGGCCTCAATGAAATGGTGCAGGCCATTCGCCTGGTGTTTGCC360               GGCCAGCGTTACATCAGCCCGCAAATTGCCCAGCAGTTGGTGTTCAAGTCATTCCAGCCT420               TCCAGTGATTCACCGTTCGATGCTTTGTCCGAGCGGGAAATCCAGATCGCGCTGATGATT480               GTCGGCTGCCAGAAAGTGCAGATCATCTCCGACAAGCTGTGCCTGTCTCCGAAAACCGTT540               AATATCTACCGTTACCGCATCTTCGAAAAGCTCTCGATCAGCAGCGATGTTGAACTGACA600               TTGCTGGCGGTTCGCCACGGCATGGTCGATGCCAGTGCCTGA642                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGCGGAGTATACCATAAG18                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATAAGCTTACCACCAGCATCGTAC24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1005 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: single                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1005                                                         (D) OTHER INFORMATION: /product="alternate sigma factor                       RpoS"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGGCTCTCAGTAAAGAAGTGCCGGAGTTTGACATCGACGATGAAGTT48                            MetAlaLeuSerLysGluValProGluPheAspIleAspAspGluVal                              151015                                                                        CTCCTTATGGAGACGGATATCGCCGCTGATTCGATGTCGAATGAGGGA96                            LeuLeuMetGluThrAspIleAlaAlaAspSerMetSerAsnGluGly                              202530                                                                        TCTGCTGTACCTTCAGTTCGTGCCAAATCCAAACACTCCGCTTCATTG144                           SerAlaValProSerValArgAlaLysSerLysHisSerAlaSerLeu                              354045                                                                        AAACAACATAAATACATTGATTACACGCGGGCGCTCGACGCGACGCAG192                           LysGlnHisLysTyrIleAspTyrThrArgAlaLeuAspAlaThrGln                              505560                                                                        TTGTATCTCAATGAAATCGGCTTTTCCCCATTGCTCTCTCCGGAAGAA240                           LeuTyrLeuAsnGluIleGlyPheSerProLeuLeuSerProGluGlu                              65707580                                                                      GAAGTTCATTTTGCGCGTCTTTCACAAAGTGGTGATCCGGCCGGGCGC288                           GluValHisPheAlaArgLeuSerGlnSerGlyAspProAlaGlyArg                              859095                                                                        AAGCGCATGATTGAAAGCAACCTGCGTCTGGTGGTGAAAATCACCCGA336                           LysArgMetIleGluSerAsnLeuArgLeuValValLysIleThrArg                              100105110                                                                     CGCTATGTCAATCGCGGGCTTTCACTGCTGGACCTCATCGAAGAGGGC384                           ArgTyrValAsnArgGlyLeuSerLeuLeuAspLeuIleGluGluGly                              115120125                                                                     AACCTCGGTNTGATCCGGNCGGTGGAGAAGATTGACCCGGAGCGCGGT432                           AsnLeuGlyXaaIleArgXaaValGluLysIleAspProGluArgGly                              130135140                                                                     TTCCGCTTCTCGACCTATGCCACCTGGTGGATCCGTCAAACCATCGAA480                           PheArgPheSerThrTyrAlaThrTrpTrpIleArgGlnThrIleGlu                              145150155160                                                                  CGGNCAATCATGAACCAGACCCGGACTATCCGNCTGCCGATTCATGTG528                           ArgXaaIleMetAsnGlnThrArgThrIleXaaLeuProIleHisVal                              165170175                                                                     GTCAAAGAGCTCAACGTCTACCTGCGGGCAGCACGTGAGCTGACTCAG576                           ValLysGluLeuAsnValTyrLeuArgAlaAlaArgGluLeuThrGln                              180185190                                                                     AAACTCGACCATGAACCTTCCCCTGNAGAAATCGCCAACCTGCTGGAG624                           LysLeuAspHisGluProSerProXaaGluIleAlaAsnLeuLeuGlu                              195200205                                                                     AAACCGGTAGGTGAGGTCAAGCGCATGCTGGGTCTCAATGAGCGGGTG672                           LysProValGlyGluValLysArgMetLeuGlyLeuAsnGluArgVal                              210215220                                                                     TCTTCAGTCGACGTCTCGCTGGGTCCGGATTCGGATAAAACCCTGCTG720                           SerSerValAspValSerLeuGlyProAspSerAspLysThrLeuLeu                              225230235240                                                                  GACACCCTCACCGACGATCGCCCAACCGATCCGTGCGAGCTGCTGCAG768                           AspThrLeuThrAspAspArgProThrAspProCysGluLeuLeuGln                              245250255                                                                     GATGACGACCTGTCGCAAAGCATCGATCAGTGGCTTTCCGAACTGACC816                           AspAspAspLeuSerGlnSerIleAspGlnTrpLeuSerGluLeuThr                              260265270                                                                     GACAAGCAGCGTGAAGTAGTGGTTCGCCGCTTCGGCTTGCGCGGCCAT864                           AspLysGlnArgGluValValValArgArgPheGlyLeuArgGlyHis                              275280285                                                                     GAAAGCAGCACCCTGGAAGATGTGGGCCTGGAGATCGGTCTTACCCGA912                           GluSerSerThrLeuGluAspValGlyLeuGluIleGlyLeuThrArg                              290295300                                                                     GAGCGGGTACGCCAGATCCAGGTCGAAGGTCTCAAGCGCCTGCGCGAG960                           GluArgValArgGlnIleGlnValGluGlyLeuLysArgLeuArgGlu                              305310315320                                                                  ATCCTCGAAAAGAACGGCCTTTCCAGCGAGTCGCTGTTCCAGTAA1005                             IleLeuGluLysAsnGlyLeuSerSerGluSerLeuPheGln*                                   325330335                                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 334 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetAlaLeuSerLysGluValProGluPheAspIleAspAspGluVal                              151015                                                                        LeuLeuMetGluThrAspIleAlaAlaAspSerMetSerAsnGluGly                              202530                                                                        SerAlaValProSerValArgAlaLysSerLysHisSerAlaSerLeu                              354045                                                                        LysGlnHisLysTyrIleAspTyrThrArgAlaLeuAspAlaThrGln                              505560                                                                        LeuTyrLeuAsnGluIleGlyPheSerProLeuLeuSerProGluGlu                              65707580                                                                      GluValHisPheAlaArgLeuSerGlnSerGlyAspProAlaGlyArg                              859095                                                                        LysArgMetIleGluSerAsnLeuArgLeuValValLysIleThrArg                              100105110                                                                     ArgTyrValAsnArgGlyLeuSerLeuLeuAspLeuIleGluGluGly                              115120125                                                                     AsnLeuGlyXaaIleArgXaaValGluLysIleAspProGluArgGly                              130135140                                                                     PheArgPheSerThrTyrAlaThrTrpTrpIleArgGlnThrIleGlu                              145150155160                                                                  ArgXaaIleMetAsnGlnThrArgThrIleXaaLeuProIleHisVal                              165170175                                                                     ValLysGluLeuAsnValTyrLeuArgAlaAlaArgGluLeuThrGln                              180185190                                                                     LysLeuAspHisGluProSerProXaaGluIleAlaAsnLeuLeuGlu                              195200205                                                                     LysProValGlyGluValLysArgMetLeuGlyLeuAsnGluArgVal                              210215220                                                                     SerSerValAspValSerLeuGlyProAspSerAspLysThrLeuLeu                              225230235240                                                                  AspThrLeuThrAspAspArgProThrAspProCysGluLeuLeuGln                              245250255                                                                     AspAspAspLeuSerGlnSerIleAspGlnTrpLeuSerGluLeuThr                              260265270                                                                     AspLysGlnArgGluValValValArgArgPheGlyLeuArgGlyHis                              275280285                                                                     GluSerSerThrLeuGluAspValGlyLeuGluIleGlyLeuThrArg                              290295300                                                                     GluArgValArgGlnIleGlnValGluGlyLeuLysArgLeuArgGlu                              305310315320                                                                  IleLeuGluLysAsnGlyLeuSerSerGluSerLeuPheGln                                    325330                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated gene activating element comprising anucleotide sequence capable of inducing the expression of at least onegene in a Pseudomonad selected for transformation, wherein said gene islatent or expressed at low levels in said Pseudomonad, and wherein saidnucleotide sequence is an rpoS sequence isolated from Pseudomonasfluorescens.
 2. The gene activating element of claim 1, wherein saidrpoS sequence encodes an RpoS product having an amino acid sequencedefined by that set forth in SEQ ID NO:
 9. 3. The gene activatingelement of claim 1, wherein said nucleotide sequence comprises the rpoSsequence shown in SEQ ID NO:
 8. 4. An isolated DNA molecule that encodesa sigma factor having an amino acid sequence defined by that set forthin SEQ ID NO:
 9. 5. The DNA molecule of claim 4, which comprises anucleotide sequence defined by that set forth in SEQ ID NO:
 8. 6. Achimeric expression construct comprising the gene activating element ofclaim
 1. 7. A chimeric expression construct comprising the DNA moleculeof claim
 4. 8. A plasmid vector comprising the gene activating elementof claim
 1. 9. A plasmid vector comprising the DNA molecule of claim 4.10. A transgenic Pseudomonad into which the gene activating element ofclaim 1 has been introduced.
 11. A transgenic Pseudomonad into which theDNA molecule of claim 4 has been introduced.
 12. A method for activatingexpression in a Pseudomonas host of at least one gene that is latent ornatively expressed at low levels, comprising introducing into said hostat least one copy of the gene activating element of claim
 1. 13. Amethod for activating expression in a Pseudomonas host of at least onegene that is latent or natively expressed at low levels, comprisingintroducing into said host at least one copy of the DNA molecule ofclaim
 4. 14. A method for activating expression in a Pseudomonas host ofat least one gene that is latent or natively expressed at low levels,comprising introducing into said host at least one copy of a DNAmolecule that encodes a Pseudomonas RpoS sigma factor.
 15. A method forrendering a Pseudomonas host effective against fungal pathogens,comprising introducing into said host at least one copy of the geneactivating element of claim
 1. 16. A method for rendering a Pseudomonashost effective against fungal pathogens, comprising introducing intosaid host at least one copy of the DNA molecule of claim 4.